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Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds

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  • Carboxypeptidase A(CPA) has a great potential application in the food and pharmaceutical industry due to its capability to hydrolyze ochratoxin A(OTA) and remove the bitterness of peptide. However, CPA is a mesophilic enzyme that cannot adequately exert its catalytic activity at elevated temperatures, which seriously restricts its industrial application. In this study, the rational design of disulfide bonds was introduced to improve the thermostability of CPA. The highly flexible regions of CPA were predicted through the HotSpot Wizard program and molecular dynamics (MD) simulations. Then, DbD and MODIP online servers were conducted to predict potential residue pairs for introducing disulfide bonds in CPA. After the conservativeness analysis of the PSSM matrix and the structural analysis of the MD simulation, two mutants with potentially enhanced thermostability were screened. Results showed that these mutants D93C/F96C and K153C/S251C compared to the wild-type(WT) exhibited increase by 10 and 10 °C in Topt, 3.4 and 2.7 min in t1/2 at 65 °C, in addition to rise of 8.5 and 11.4 °C in T5015, respectively. Furthermore, the molecular mechanism responsible for thermostability was investigated from the perspective of advanced structure and molecular interactions. The enhanced thermostability of both mutants was not only associated with the more stable secondary structure and the introduction of disulfide bonds but also related to the changes in hydrogen bonds and the redistribution of surface charges in mutant regions. This study showed for the first time that the rational design of disulfide bonds is an effective strategy to enhance the thermostability of CPA, providing in this way a broader industrial application.
  • Crops require a variety of nutrients for growth and nitrogen is particularly important. Nitrogen is the primary factor limiting plant growth and yield formation, and it also plays a significant role in improving product quality[14]. Nitrogen accounts for 1%−3% of the dry weight of plants and is a component of many compounds. For example, it is an important part of proteins, a component of nucleic acids, the skeleton of cell membranes, and a constituent of chlorophyll[5,6]. When the plant is deficient in nitrogen, the synthesis process of nitrogen-containing substances such as proteins decrease significantly, cell division and elongation are restricted, and chlorophyll content decreases, and this leads to short and thin plants, small leaves, and pale color[2,7,8]. If nitrogen in the plant is in excess, a large number of carbohydrates will be used for the synthesis of proteins, chlorophyll, and other substances, so that cells are large and thin-walled, and easy to be attacked by pests and diseases. At the same time, the mechanical tissues in the stem are not well developed and are prone to collapse[3,8,9]. Therefore, the development of new crop varieties with both high yields and improved nitrogen use efficiency (NUE) is an urgently needed goal for more sustainable agriculture with minimal nitrogen demand.

    Plants obtain inorganic nitrogen from the soil, mainly in the form of NH4+ and nitrate (NO3)[1013]. Nitrate uptake by plants occurs primarily in aerobic environments[3]. Transmembrane proteins are required for nitrate uptake from the external environment as well as for transport and translocation between cells, tissues, and organs. NITRATE TRANSPORTER PROTEIN 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family (NPF), NRT2, CHLORIDE CHANNEL (CLC) family, and SLOW ACTIVATING ANION CHANNEL are four protein families involved in nitrate transport[14]. One of the most studied of these is NRT1.1, which has multiple functions[14]. NRT1.1 is a major nitrate sensor, regulating many aspects of nitrate physiology and developmental responses, including regulating the expression levels of nitrate-related genes, modulating root architecture, and alleviating seed dormancy[1518].

    There is mounting evidence that plant growth and development are influenced by interactions across numerous phytohormone signaling pathways, including abscisic acid, gibberellins, growth hormones, and cytokinins[3,19,20]. To increase the effectiveness of plant nitrogen fertilizer application, it may be possible to tweak the signaling mediators or vary the content of certain phytohormones. Since the 1930s, research on the interplay between growth factors and N metabolism has also been conducted[3]. The Indole acetic acid (IAA) level of plant shoots is shown to decrease in early studies due to N shortage, although roots exhibit the reverse tendency[3,21]. In particular, low NO3 levels caused IAA buildup in the roots of Arabidopsis, Glycine max, Triticum aestivum, and Zea mays, indicating that IAA is crucial for conveying the effectiveness of exogenous nitrogen to the root growth response[20,22,23].

    Studies have shown that two families are required to control the expression of auxin-responsive genes: one is the Auxin Response Factor (ARF) and the other is the Aux/IAA repressor family[2426]. As the transcription factor, the ARF protein regulates the expression of auxin response genes by specifically binding to the TGTCNN auxin response element (AuxRE) in promoters of primary or early auxin response genes[27]. Among them, rice OsARF18, as a class of transcriptional repressor, has been involved in the field of nitrogen utilization and yield[23,28]. In rice (Oryza sativa), mutations in rice salt tolerant 1 (rst1), encoding the OsARF18 gene, lead to the loss of its transcriptional repressor activity and up-regulation of OsAS1 expression, which accelerates the assimilation of NH4+ to Asn and thus increases N utilization[28]. In addition, dao mutant plants deterred the conversion of IAA to OxIAA, thus high levels of IAA strongly activates OsARF18, which subsequently represses the expression of OsARF2 and OsSUT1 by directly binding to the AuxRE and SuRE promoter motifs, resulting in the inhibition of carbohydrate partitioning[23]. As a result, rice carrying the dao has low yields.

    Apples (Malus domestica) are used as a commercially important crop because of their high ecological adaptability, high nutritional value, and annual availability of fruit[29]. To ensure high apple yields, growers promote rapid early fruit yield growth by applying nitrogen. However, the over-application of nitrogen fertilizer to apples during cultivation also produces common diseases and the over-application of nitrogen fertilizer is not only a waste of resources but also harmful to the environment[29]. Therefore, it is of great significance to explore efficient nitrogen-regulated genes to understand the uptake and regulation of nitrogen fertilizer in apples, and to provide reasonable guidance for nitrogen application during apple production[30]. In this study, MdARF18 is identified which is a key transcription factor involved in nitrate uptake and transport in apples and MdARF18 reduces NO3 uptake and assimilation. Further analysis suggests that MdRF18 may inhibit the transcriptional level of MdNRT1.1 promoter by directly binding to its TGTCTT target, thus affecting normal plant growth.

    The protein sequence of apple MdARF18 (MD07G1152100) was obtained from The Apple Genome (https://iris.angers.inra.fr/gddh13/). Mutant of arf18 (GABI_699B09) sequence numbers were obtained from the official TAIR website (www.arabidopsis.org). The protein sequences of ARF18 from different species were obtained from the protein sequence of apple MdARF18 on the NCBI website. Using these data, a phylogenetic tree with reasonably close associations was constructed[31].

    Protein structural domain prediction of ARF18 was performed on the SMART website (https://smart.embl.de/). Motif analysis of ARF18 was performed by MEME (https://meme-suite.org/meme/tools/meme). Clustal was used to do multiple sequence comparisons. The first step was accessing the EBI web server through the Clustal Omega channel. The visualization of the results was altered using Jalview, which may be downloaded from www.jalview.org/download.[32]

    The apple 'Orin' callus was transplanted on MS medium containing 1.5 mg·L−1 6-benzylaminopurine (6-BA) and 0.5 mg·L−1 2,4 dichlorophenoxyacetic acid (2,4-D) at 25 °C, in the dark, at 21-d intervals. 'Royal Gala' apple cultivars were cultured in vermiculite and transplanted at 25 °C every 30 d. The Arabidopsis plants used were of the Columbia (Col-0) wild-type variety. Sowing and germinating Arabidopsis seeds on MS nutrient medium, and Arabidopsis seeds were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h)[33].

    The nutrient solution in the base contained 1.0 mM CaCl2, 1.0 mM KH2PO4, 1.0 mM MgSO4, 0.1 mM FeSO4·7H2O 0.1 mM Na2EDTA·2H2O, 50 μM MnSO4·H2O, 50 μM H3BO3, 0.05 μM CuSO4·5H2O, 0.5 μM Na2MoO4·2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl· 6H2O. 2H2O, 15 μM ZnSO4·7H2O, 2.5 μM KI and 0.05 μM CoCl·6H2O, and 0.05 μM CoCl·6H2O, supplemented with 0.5 mM, 2 mM, and 10 mM KNO3 as the sole nitrogen source, and added with the relevant concentrations of KCl to maintain the same K concentration[33,34].

    For auxin treatment, 12 uniformly growing apple tissue-cultured seedlings (Malus domestica 'Royal Gala') were selected from each of the control and treatment groups, apple seedlings were incubated in a nutrient solution containing 1.5 mg·L−1 6-BA, 0.2 mg·L−1 naphthalene acetic acid, and IAA (10 μM) for 50 d, and then the physiological data were determined. Apple seedlings were incubated and grown at 25 °C (light/dark cycle of 16 h/8 h).

    For nitrate treatment, Arabidopsis seedlings were transferred into an MS medium (containing different concentrations of KNO3) as soon as they germinated to test root development. Seven-day-old Arabidopsis were transplanted into vermiculite and then treated with a nutrient solution containing different concentrations of KNO3 (0.5, 2, 10 mM) and watered at 10-d intervals. Apple calli were treated with medium containing 1.5 mg·L−1 6-BA, 0.5 mg·L−1 2,4-D, and varying doses of KNO3 (0.5, 2, and 10 mM) for 25 d, and samples were examined for relevant physiological data. Apple calli were subjected to the same treatment for 1 d for GUS staining[35].

    To obtain MdARF18 overexpression materials, the open reading frame (ORF) of MdARF18 was introduced into the pRI-101 vector. To obtain pMdNRT1.1 material, the 2 kb segment located before the transcription start site of MdNRT1.1 was inserted into the pCAMBIA1300 vector. The Agrobacterium tumefaciens LBA4404 strain was cultivated in lysozyme broth (LB) medium supplemented with 50 mg·L−1 kanamycin and 50 mg·L−1 rifampicin. The MdARF18 overexpression vector and the ProMdNRT1.1::GUS vector were introduced into Arabidopsis and apple callus using the flower dip transformation procedure. The third-generation homozygous transgenic Arabidopsis (T3) and transgenic calli were obtained[36]. Information on the relevant primers designed is shown in Supplemental Table S1.

    Plant DNA and RNA were obtained using the Genomic DNA Kit and the Omni Plant RNA Kit (tDNase I) (Tiangen, Beijing, China)[37].

    cDNA was synthesized for qPCR by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). The cDNA for qPCR was synthesized by using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Dalian, China). Quantitative real-time fluorescence analysis was performed by using the UltraSYBR Mixture (Low Rox) kit (ComWin Biotech Co. Ltd., Beijing, China). qRT-PCR experiments were performed using the 2−ΔΔCᴛ method for data analysis. The data were analyzed by the 2−ΔΔCᴛ method[31].

    GUS staining buffer contained 1 mM 5-bromo-4-chloro-3-indolyl-β-glutamic acid, 0.01 mM EDTA, 0.5 mM hydrogen ferrocyanide, 100 mM sodium phosphate (pH 7.0), and 0.1% (v/v) Triton X-100 was maintained at 37 °C in the dark. The pMdNRT1.1::GUS construct was transiently introduced into apple calli. To confirm whether MdNRT1.1 is activated or inhibited by MdARF18, we co-transformed 35S::MdARF18 into pMdNRT1.1::GUS is calling. The activity of transgenic calli was assessed using GUS labeling and activity assays[33,38].

    The specimens were crushed into fine particles, combined with 1 mL of ddH2O, and thereafter subjected to a temperature of 100 °C for 30 min. The supernatant was collected in a flow cell after centrifugation at 12,000 revolutions per minute for 10 min. The AutoAnalyzer 3 continuous flow analyzer was utilized to measure nitrate concentrations. (SEAL analytical, Mequon, WI, USA). Nitrate reductase (NR) activity was characterized by the corresponding kits (Solarbio Life Science, Beijing, China) using a spectrophotometric method[31].

    Y1H assays were performed as previously described by Liu et al.[39]. The coding sequence of MdARF18 was integrated into the pGADT7 expression vector, whereas the promoter region of MdNRT1.1 was included in the pHIS2 reporter vector. Subsequently, the constitutive vectors were co-transformed into the yeast monohybrid strain Y187. The individual transformants were assessed on a medium lacking tryptophan, leucine, and histidine (SDT/-L/-H). Subsequently, the positive yeast cells were identified using polymerase chain reaction (PCR). The yeast strain cells were diluted at dilution factors of 10, 100, 1,000, and 10,000. Ten μL of various doses were added to selective medium (SD-T/-L/-H) containing 120 mM 3-aminotriazole (3-AT) and incubated at 28 °C for 2−3 d[37].

    Dual-luciferase assays were performed as described previously[40]. Full-length MdARF18 was cloned into pGreenII 62-SK to produce MdARF18-62-SK. The promoter fragment of MdNRT1.1 was cloned into pGreenII 0800-LUC to produce pMdNRT1.1-LUC. Different combinations were transformed into Agrobacterium tumefaciens LBA4404 and the Agrobacterium solution was injected onto the underside of the leaves of tobacco (Nicotiana benthamiana) leaves abaxially. The Dual Luciferase Reporter Kit (Promega, www.promega.com) was used to detect fluorescence activity.

    Total protein was extracted from wild-type and transgenic apple calli with or without 100 μM MG132 treatment. The purified MdARF18-HIS fusion protein was incubated with total protein[41]. Samples were collected at the indicated times (0, 1, 3, 5, and 7 h).

    Protein gel blots were analyzed using GST antibody. ACTIN antibody was used as an internal reference. All antibodies used in this study were provided by Abmart (www.ab-mart.com).

    Unless otherwise noted, every experiment was carried out independently in triplicate. A one-way analysis of variance (ANOVA) was used to establish the statistical significance of all data, and Duncan's test was used to compare results at the p < 0.05 level[31].

    To investigate whether auxin affects the effective uptake of nitrate in apple, we first externally applied IAA under normal N (5 mM NO3) environment, and this result showed that the growth of Gala apple seedlings in the IAA-treated group were better than the control, and their fresh weights were heavier than the control group (Fig. 1a, d). The N-related physiological indexes of apple seedlings also showed that the nitrate content and NR activity of the root part of the IAA-treated group were significantly higher than the control group, while the nitrate content and NR activity of the shoot part were lower than the control group (Fig. 1b, c). These results demonstrate that auxin could promote the uptake of nitrate and thus promotes growth of plants.

    Figure 1.  Auxin enhances nitrate uptake of Gala seedlings. (a) Phenotypes of apple (Malus domestica 'Royal Gala') seedlings grown nutritionally for 50 d under IAA (10 μM) treatment. (b) Nitrate content of shoot and root apple (Malus domestica 'Royal Gala') seedlings treated with IAA. (c) NR activity in shoot and root of IAA treatment apple (Malus domestica 'Royal Gala') seedlings. (d) Seedling fresh weight under IAA treatment. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).

    To test whether auxin affects the expression of genes related to nitrogen uptake and metabolism. For the root, the expression levels of MdNRT1.1, MdNRT2.1, MdNIA1, MdNIA2, and MdNIR were higher than control group (Supplemental Fig. S1a, f, hj), while the expression levels of MdNRT1.2, MdNRT1.6 and MdNRT2.5 were lower than control group significantly (Supplemental Fig. S1b, d, g). For the shoot, the expression of MdNRT1.1, MdNRT1.5, MdNRT1.6, MdNRT1.7, MdNRT2.1, MdNRT2.5, MdNIA1, MdNIA2, and MdNIR genes were significantly down-regulated (Supplemental Fig. S1a, cj). This result infers that the application of auxin could mediate nitrate uptake in plants by affecting the expression levels of relevant nitrate uptake and assimilation genes.

    Since the auxin signaling pathway requires the regulation of the auxin response factors (ARFs)[25,27], it was investigated whether members of ARF genes were nitrate responsive. Firstly, qPCR quantitative analysis showed that the five subfamily genes of MdARFs (MdARF9, MdARF2, MdARF12, MdARF3, and MdARF18) were expressed at different levels in various organs of the plant (Supplemental Fig. S2). Afterward, the expression levels of five ARF genes were analyzed under different concentrations of nitrate treatment (Fig. 2), and it was concluded that these genes represented by each subfamily responded in different degrees, but the expression level of MdARF18 was up-regulated regardless of low or high nitrogen (Fig. 2i, j), and the expression level of MdARF18 showed a trend of stable up-regulation under IAA treatment (Supplemental Fig. S3). The result demonstrates that MdARFs could affect the uptake of external nitrate by plants and MdARF18 may play an important role in the regulation of nitrate uptake.

    Figure 2.  Relative expression analysis of MdARFs subfamilies in response to different concentrations of nitrate. Expression analysis of representative genes from five subfamilies of MdARF transcription factors. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).

    MdARF18 (MD07G1152100) was predicted through The Apple Genome website (https://iris.angers.inra.fr/gddh13/) and it had high fitness with AtARF18 (AT3G61830). The homologs of ARF18 from 15 species were then identified in NCBI (www.ncbi.nlm.nih.gov) and then constructed an evolutionary tree (Supplemental Fig. S4). The data indicates that MdARF18 was most closely genetically related to MbARF18 (Malus baccata), indicating that they diverged recently in evolution (Supplemental Fig. S4). Conserved structural domain analyses indicated that all 15 ARF18 proteins had highly similar conserved structural domains (Supplemental Fig. S5). In addition, multiple sequence alignment analysis showed that all 15 ARF18 genes have B3-type DNA-binding domains (Supplemental Fig. S6), which is in accordance with the previous reports on ARF18 protein structure[26].

    To explore whether MdARF18 could affect the development of the plant's root system. Firstly, MdARF18 was heterologously expressed into Arabidopsis, and an arf18 mutant (GABI_699B09) Arabidopsis was also obtained (Supplemental Fig. S7). Seven-day-old MdARF18 transgenic Arabidopsis and arf18 mutants were treated in a medium with different nitrate concentrations for 10 d (Fig. 3a, b). After observing results, it was found that under the environment of high nitrate concentration, the primary root of MdARF18 was shorter than arf18 and wild type (Fig. 3c), and the primary root length of arf18 is the longest (Fig. 3c), while there was no significant difference in the lateral root (Fig. 3d). For low nitrate concentration, there was no significant difference in the length of the primary root, and the number of lateral roots of MdARF18 was slightly more than wild type and arf18 mutant. These results suggest that MdARF18 affects root development in plants. However, in general, low nitrate concentrations could promote the transport of IAA by NRT1.1 and thus inhibit lateral root production[3], so it might be hypothesized that MdARF18 would have some effect on MdNRT1.1 thus leading to the disruption of lateral root development.

    Figure 3.  MdARF18 inhibits root development. (a) MdARF18 inhibits root length at 10 mM nitrate concentration. (b) MdARF18 promotes lateral root growth at 0.5 mM nitrate concentration. (c) Primary root length statistics. (d) Lateral root number statistics. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).

    To investigate whether MdARF18 affects the growth of individual plants under different concentrations of nitrate, 7-day-old overexpression MdARF18, and arf18 mutants were planted in the soil and incubated for 20 d. It was found that arf18 had the best growth of shoot, while MdARF18 had the weakest shoot growth at any nitrate concentration (Fig. 4a). MdARF18 had the lightest fresh weight and the arf18 mutant had the heaviest fresh weight (Fig. 4b). N-related physiological indexes revealed that the nitrate content and NR activity of arf18 were significantly higher than wild type, whereas MdARF18 materials were lower than wild type (Fig. 4c, d). More detail, MdARF18 had the lightest fresh weight under low and normal nitrate, while the arf18 mutant had the heaviest fresh weight, and the fresh weight of arf18 under high nitrate concentration did not differ much from the wild type (Fig. 4b). Nitrogen-related physiological indexes showed that the nitrate content of arf18 was significantly higher than wild type, while MdARF18 was lower than wild type. The NR activity of arf18 under high nitrate did not differ much from the wild type, but the NR activity of MdARF18 was the lowest in any treatment (Fig. 4c, d). These results indicate that MdARF18 significantly inhibits plant growth by inhibiting plants to absorb nitrate, and is particularly pronounced at high nitrate concentrations.

    Figure 4.  Ectopic expression of MdARF18 inhibits Arabidopsis growth. (a) Status of Arabidopsis growth after one month of incubation at different nitrate concentrations. (b) Fresh weight of Arabidopsis. (c) Nitrate content of Arabidopsis. (d) NR activity in Arabidopsis. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).

    In addition, to further validate this conclusion, MdARF18 overexpression calli were obtained and treated with different concentrations of nitrate (Supplemental Fig. S8). The results show that the growth of overexpressed MdARF18 was weaker than wild type in both treatments (Supplemental Fig. S9a). The fresh weight of MdARF18 was significantly lighter than wild type (Supplemental Fig. S9b), and its nitrate and NR activity were lower than wild type (Supplemental Fig. S9c, d), which was consistent with the above results (Fig. 4). This result further confirms that MdARF18 could inhibit the development of individual plants by inhibiting the uptake of nitrate.

    Nitrate acts as a signaling molecule that takes up nitrate by activating the NRT family as well as NIAs and NIR[3,34]. To further investigate the pathway by which MdARF18 inhibits plant growth and reduces nitrate content, qRT-PCR was performed on the above plant materials treated with different concentrations of nitrate (Fig. 5). The result shows that the expression levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR were all down-regulated in overexpression of MdARF18, and up-regulated in the arf18 mutant (Fig. 5a, hj). There was no significant change in AtNRT1.2 at normal nitrate levels, but AtNRT1.2 expression levels were down-regulated in MdARF18 and up-regulated in arf18 at both high and low nitrate levels (Fig. 5b). This trend in the expression levels of these genes might be consistent with the fact that MdARF18 inhibits the expression of nitrogen-related genes and restricts plant growth. The trend in the expression levels of these genes is consistent with MdARF18 restricting plant growth by inhibiting the expression of nitrogen-related genes. However, AtNRT1.5, AtNRT1.6, AtNRT1.7, AtNRT2.1, and AtNRT2.5 did not show suppressed expression levels in MdARF18 (Fig. 5cg). These results suggest that MdARF18 inhibits nitrate uptake and plant growth by repressing some of the genes for nitrate uptake or assimilation.

    Figure 5.  qPCR-RT analysis of N-related genes. Expression analysis of N-related genes in MdARF18 transgenic Arabidopsis at different nitrate concentrations. Bars represent the mean ± SD (n = 3). Different letters above the bars indicate significant differences using the LSD test (p < 0.05).

    In addition, to test whether different concentrations of nitrate affect the protein stability of MdARF18. However, it was found that there was no significant difference in the protein stability of MdARF18 at different concentrations of nitrate (Supplemental Fig. S10). This result suggests that nitrate does not affect the degradation of MdARF18 protein.

    To further verify whether MdARF18 can directly bind N-related genes, firstly we found that the MdNRT1.1 promoter contains binding sites to ARF factors (Fig. 6a). The yeast one-hybrid research demonstrated an interaction between MdARF18 and the MdNRT1.1 promoter, as shown in Fig. 6b. Yeast cells that were simultaneously transformed with MdNRT1.1-P-pHIS and pGADT7 were unable to grow in selected SD medium. However, cells that were transformed with MdNRT1.1-P-pHIS and MdARF18-pGADT7 grew successfully in the selective medium. The result therefore hypothesizes that MdARF18 could bind specifically to MdNRT1.1 promoter to regulate nitrate uptake in plants.

    Figure 6.  MdARF18 binds directly to the promoter of MdNRT1.1. (a) Schematic representation of MdNRT1.1 promoter. (b) Y1H assay of MdARF18 bound to the MdNRT1.1 promoter in vitro. 10−1, 10−2, 10−3, and 10−4 indicate that the yeast concentration was diluted 10, 100, 1,000, and 10,000 times, respectively. 3-AT stands for 3-Amino-1,2,4-triazole. (c) Dual luciferase assays demonstrate the binding of MdARF18 with MdNRT1.1 promoter. The horizontal bar on the left side of the right indicates the captured signal intensity. Empty LUC and 35S vectors were used as controls. Representative images of three independent experiments are shown here.

    To identify the inhibition or activation of MdNRT1.1 by MdARF18, we analyzed their connections by Dual luciferase assays (Fig. 6c), and also analyzed the fluorescence intensity (Supplemental Fig. S11). It was concluded that the fluorescence signals of cells carrying 35Spro and MdNRT1.1pro::LUC were stronger, but the mixture of 35Spro::MdARF18 and MdNRT1.1pro::LUC injected with fluorescence signal intensity was significantly weakened. Next, we transiently transformed the 35S::MdARF18 into pMdNRT1.1::GUS transgenic calli (Fig. 7). GUS results first showed that the color depth of pMdNRT1.1::GUS and 35S::MdARF18 were significantly lighter than pMdnNRT1.1::GUS alone (Fig. 7a). GUS enzyme activity, as well as GUS expression, also indicated that the calli containing pMdnNRT1.1::GUS alone had a stronger GUS activity (Fig. 7b, c). In addition, the GUS activity of calli containing both pMdNRT1.1:GUS and 35S::MdARF18 were further attenuated under both high and low nitrate concentrations (Fig. 7a). These results suggest that MdARF18 represses MdNRT1.1 expression by directly binding to the MdNRT1.1 promoter region.

    Figure 7.  MdARF18 inhibits the expression of MdNRT1.1. (a) GUS staining experiment of pMdNRT1.1::GUS transgenic calli and transgenic calli containing both pMdNRT1.1::GUS and 35S::MdARF18 with different nitrate treatments. (b) GUS activity assays in MdARF18 overexpressing calli with different nitrate treatments. (c) GUS expression level in MdARF18 overexpressing calli with different nitrate treatments. Bars represent the mean ± SD (n = 3). Different numbers of asterisk above the bars indicate significant differences using the LSD test (*p < 0.05 and **p< 0.01).

    Plants replenish their nutrients by absorbing nitrates from the soil[42,43]. Previous studies have shown that some of the plant hormones such as IAA, GA, and ABA interact with nitrate[25,4445]. The effect of nitrate on the content and transport of IAA has been reported in previous studies, e.g., nitrate supply reduced IAA content in Arabidopsis, wheat, and maize roots and inhibited the transport of IAA from shoot to root[20,21]. In this study, it was found that auxin treatment promoted individual fresh weight gain and growth (Fig. 1a, b). Nitrate content and NR activity were also significantly higher in their root parts (Fig. 1c, d) and also affected the transcript expression levels of related nitrate uptake and assimilation genes (Supplemental Fig. S1). Possibly because IAA can affect plant growth by influencing the uptake of external nitrates by the plant.

    ARFs are key transcription factors to regulate auxin signaling[4649]. We identified five representative genes of the apple MdARFs subfamily and they all had different expression patterns (Supplemental Fig. S2). The transcript levels of each gene were found to be affected to different degrees under different concentrations of nitrate, but the expression level of MdARF18 was up-regulated under both low and high nitrate conditions (Fig. 2). The transcript level of MdARF18 was also activated under IAA treatment (Supplemental Fig. S3), so MdARF18 began to be used in the study of the mechanism of nitrate uptake in plants. In this study, an Arabidopsis AtARF18 homolog was successfully cloned and named MdARF18 (Supplemental Figs S4, S5). It contains a B3-type DNA-binding structural domain consistent with previous studies of ARFs (Supplemental Fig. S6), and arf18 mutants were also obtained and their transcript levels were examined (Supplemental Fig. S7).

    Plants rely on rapid modification of the root system to efficiently access effective nitrogen resources in the soil for growth and survival. The plasticity of root development is an effective strategy for accessing nitrate, and appropriate concentrations of IAA can promote the development of lateral roots[7,44]. The present study found that the length of the primary root was shortened and the number of lateral roots did increase in IAA-treated Gl3 apple seedlings (Supplemental Fig. S12). Generally, an environment with low concentrations of nitrate promotes the transport of IAA by AtNRT1.1, which inhibits the growth of lateral roots[14]. However, in the research of MdARF18 transgenic Arabidopsis, it was found that the lateral roots of MdARF18-OX increased under low concentrations of nitrate, but there was no significant change in the mutant arf18 (Fig. 3). Therefore, it was hypothesized that MdARF18 might repress the expression of the MdNRT1.1 gene or other related genes that can regulate root plasticity, thereby affecting nitrate uptake in plants.

    In rice, several researchers have demonstrated that OsARF18 significantly regulates nitrogen utilization. Loss of function of the Rice Salt Tolerant 1 (RST1) gene (encoding OsARF18) removes its ability to transcriptionally repress OsAS1, accelerating the assimilation of NH4+ to Asn and thereby increasing nitrogen utilization[28]. During soil incubation of MdARF18-OX Arabidopsis, it was found that leaving aside the effect of differences in nitrate concentration, the arf18 mutant grew significantly better than MdARF18-OX and had higher levels of nitrate and NR activity in arf18 than in MdARF18-OX. This demonstrates that MdARF18 may act as a repressor of nitrate uptake and assimilation, thereby inhibiting normal plant development (Fig. 4). Interestingly, an adequate nitrogen environment promotes plant growth, but MdARF18-OX Arabidopsis growth and all physiological indexes were poorer under high nitrate concentration than MdARF18-OX at other concentrations. We hypothesize that MdARF18 may be activated more intensively at high nitrate concentrations, or that MdARF18 suppresses the expression levels of genes for nitrate uptake or assimilation (genes that may play a stronger role at high nitrate concentrations), thereby inhibiting plant growth. In addition, we obtained MdARF18 transgenic calli (Supplemental Fig. S8) and subjected them to high and low concentrations of nitrate, and also found that MdARF18 inhibited the growth of individuals at both concentrations (Supplemental Fig. S9). This further confirms that MdARF18 inhibits nitrate uptake in individuals.

    ARF family transcription factors play a key role in transmitting auxin signals to alter plant growth and development, e.g. osarf1 and osarf24 mutants have reduced levels of OsNRT1.1B, OsNRT2.3a and OsNIA2 transcripts[22]. Therefore, further studies are needed to determine whether MdARF18 activates nitrate uptake through different molecular mechanisms. The result revealed that the transcript levels of AtNRT1.1, AtNIA1, AtNIA2, and AtNIR in MdARF18-OX were consistent with the developmental pattern of impaired plant growth (Fig. 5). Unfortunately, we attempted to explore whether variability in nitrate concentration affects MdARF18 to differ at the protein level, but the two did not appear to differ significantly (Supplemental Fig. S10).

    ARF transcription factors act as trans-activators/repressors of N metabolism-related genes by directly binding to TGTCNN/NNGACA-containing fragments in the promoter regions of downstream target genes[27,50]. The NRT family plays important roles in nitrate uptake, transport, and storage, and NRT1.1 is an important dual-affinity nitrate transporter protein[7,5052], and nitrogen utilization is very important for apple growth[53,54]. We identified binding sites in the promoters of these N-related genes that are compatible with ARF factors, and MdARF18 was found to bind to MdNRT1.1 promoter by yeast one-hybrid technique (Fig. 6a, b). It was also verified by Dual luciferase assays that MdARF18 could act as a transcriptional repressor that inhibited the expression of the downstream gene MdNRT1.1 (Fig. 6c), which inhibited the uptake of nitrate in plants. In addition, the GUS assay was synchronized to verify that transiently expressed pMdNRT1.1::GUS calli with 35S::MdARF18 showed a lighter staining depth and a significant decrease in GUS transcript level and enzyme activity (Fig. 7). This phenomenon was particularly pronounced at high concentrations of nitrate. These results suggest that MdARF18 may directly bind to the MdNRT1.1 promoter and inhibit its expression, thereby suppressing NO3 metabolism and decreasing the efficiency of nitrate uptake more significantly under high nitrate concentrations.

    In conclusion, in this study, we found that MdARF18 responds to nitrate and could directly bind to the TGTCTT site of the MdNRT1.1 promoter to repress its expression. Our findings provide new insights into the molecular mechanisms by which MdARF18 regulates nitrate transport in apple.

    The authors confirm contribution to the paper as follows: study conception and design: Liu GD; data collection: Liu GD, Rui L, Liu RX; analysis and interpretation of results: Liu GD, Li HL, An XH; draft manuscript preparation: Liu GD; supervision: Zhang S, Zhang ZL; funding acquisition: You CX, Wang XF; All authors reviewed the results and approved the final version of the manuscript.

    Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

    This work was supported by the National Natural Science Foundation of China (32272683), the Shandong Province Key R&D Program of China (2022TZXD008-02), the China Agriculture Research System of MOF and MARA (CARS-27), the National Key Research and Development Program of China (2022YFD1201700), and the National Natural Science Foundation of China (NSFC) (32172538).

  • The authors declare that they have no conflict of interest.

  • Supplemental Fig. S1 The residue pairs forming potential disulfide bridges of carboxypeptidase A.
    Supplemental Fig. S2 The Sequence conservation of carboxypeptidase A.
    Supplemental Fig. S3 The ERRAT evaluation of mutant carboxypeptidase A D93C/F96C (a) and K153C/S251C (b).
    Supplemental Fig. S4 The Ramachandran plots of mutant carboxypeptidase A D93C/F96C (a) and K153C/S251C (b).
    Supplemental Fig. S5 Screening His+ transformations by MD plates of WT (a), D93C/F96C (b) and K153C/S251C (c).
    Supplemental Fig. S6 Screening of high-copy strains by G418 with gradient concentrations. (a) WT. (b)D93C/F96C. (c) K153C/S251C.
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    Zhang H, Zhao Z, Zhu M, Logrieco AF, Wang H, et al. 2024. Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds. Food Innovation and Advances 3(2): 191−201 doi: 10.48130/fia-0024-0017
    Zhang H, Zhao Z, Zhu M, Logrieco AF, Wang H, et al. 2024. Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds. Food Innovation and Advances 3(2): 191−201 doi: 10.48130/fia-0024-0017

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Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds

Food Innovation and Advances  3 2024, 3(2): 191−201  |  Cite this article

Abstract: Carboxypeptidase A(CPA) has a great potential application in the food and pharmaceutical industry due to its capability to hydrolyze ochratoxin A(OTA) and remove the bitterness of peptide. However, CPA is a mesophilic enzyme that cannot adequately exert its catalytic activity at elevated temperatures, which seriously restricts its industrial application. In this study, the rational design of disulfide bonds was introduced to improve the thermostability of CPA. The highly flexible regions of CPA were predicted through the HotSpot Wizard program and molecular dynamics (MD) simulations. Then, DbD and MODIP online servers were conducted to predict potential residue pairs for introducing disulfide bonds in CPA. After the conservativeness analysis of the PSSM matrix and the structural analysis of the MD simulation, two mutants with potentially enhanced thermostability were screened. Results showed that these mutants D93C/F96C and K153C/S251C compared to the wild-type(WT) exhibited increase by 10 and 10 °C in Topt, 3.4 and 2.7 min in t1/2 at 65 °C, in addition to rise of 8.5 and 11.4 °C in T5015, respectively. Furthermore, the molecular mechanism responsible for thermostability was investigated from the perspective of advanced structure and molecular interactions. The enhanced thermostability of both mutants was not only associated with the more stable secondary structure and the introduction of disulfide bonds but also related to the changes in hydrogen bonds and the redistribution of surface charges in mutant regions. This study showed for the first time that the rational design of disulfide bonds is an effective strategy to enhance the thermostability of CPA, providing in this way a broader industrial application.

    • Carboxypeptidase A (CPA) (EC 3.4.17.1) is a metallocarboxypeptidase belonging to the M14 family. It consists of 307 amino acids with a molecular weight of 34.472 kDa, which are mainly divided into two groups, CPA1 and CPA2. As an exopeptidase, CPA exclusively releases amino acids from the C-terminal region of peptides or proteins[1]. CPA1 hydrolyzes smaller aliphatic amino acids and CPA2 prefers to hydrolyze larger aromatic amino acids. In the structure of CPA, there is a Zn2+ essential for the enzyme activity[2]. It was found that CPA has significant applications in industrial fields. Firstly, CPA with excellent ochratoxin A (OTA) degradation activity hydrolyzes toxic OTA into non-toxic ochratoxin α (OTα) and L-phenylalanine[3], showing great potential for OTA degradation in the food and feed industry. In addition, CPA can not only remove the bitterness of peptides in food industries and reduce food allergies, but also be applied in manufacturing functional or fermented foods[4]. As a mesophilic enzyme, the catalytic activity of CPA tends to decrease rapidly or inactivate at elevated temperatures, seriously limiting its industrial application.

      High thermostability is always considered to be a crucial prerequisite to evaluating the feasibility of enzymes for industrial applications[5]. Typically, higher thermostability confers greater competitiveness and wider applicability to enzymes[6]. Enzymes with thermostability can maintain long-lasting catalytic activity reducing the reaction time and the production cost. However, most natural enzymes perform their catalytic function under relatively mild conditions. They are not well adapted to harsh industrial processes. Therefore, adopting appropriate strategies to improve the thermostability of biological enzymes is of great value for industrial and biomedical applications. Protein engineering, as one of the most ideal strategies to improve enzyme thermostability, mainly includes three approaches (rational design, directed evolution, and semi-rational design)[7]. Rational design is based on an understanding of the relationships between enzyme structure and function[8]. It can quickly predict target regions and modification sites with the help of computer programs. It has the advantages of a small screening workload, high prediction accuracy, and efficiency. Currently, rational design strategies successfully applied include homology comparison[9], introduction of salt bridges[10] or disulfide bonds[11], substitution of proline[12] or glycine[13]. In particular, the disulfide bond is formed between the thiol groups of two spatially close cysteine residues, which is significant for enzyme folding, stability, and function[14]. As a covalent interaction, it maintains stability of the structure of enzymes by reducing conformational entropy. It was found that the energy provided by disulfide bonds to maintain enzyme stability was greatest compared with other non-covalent interaction forces such as hydrogen bonds and salt bridges[15]. In recent years, a large number of studies have shown that the rational design of introducing disulfide bonds could realize the improvement of enzyme thermostability. For example, Zhang et al. introduced a disulfide bond at the C-terminus of B. licheniformis phytase, and successfully improved the thermostability of it[16]. In addition, transglutaminase[17], lipase[18], and ammonia lyase[19] also achieved enhanced thermostability by rational design of disulfide bonds. However, not all disulfide bond introductions were beneficial. The introduction of disulfide bonds at the N-terminus negatively affected the thermostability of the 1,3-1,4-β-glucanase mutant V59C-Y86C[20]. Therefore, it is crucial to select appropriate strategies for introducing disulfide bonds in suitable regions or sites. A previous study showed that strategically introducing disulfide bonds in flexible regions could provide stronger rigid support and create a protected microenvironment for the enzyme, effectively improving thermostability and catalytic efficiency[15].

      In our previous study, the thermostability of CPA was successfully enhanced by multiple computer-aided rational design based on amino acids preferences at β-turns while the catalytic activity was significantly decreased[21]. In this study, the rational design of disulfide bonds by HotSpot Wizard program and molecular dynamics (MD) simulations was proposed to improve the thermostability of CPA and reduce the loss of activity in ochratoxin degradation by obtaining mutants and studying the molecular mechanism responsible for thermostability.

    • The strains E. coli DH5α carrying pPIC9K plasmids and P. pastoris GS115 were stored at our lab. The restriction enzyme Sac I was purchased from Takara Biotechnology Co., Ltd. (Beijing, China). The SDS-PAGE fast protein gel kit was purchased from Dakewe Biotechnology Co., Ltd. (Beijing, China). Detergent Compatible Bradford Protein Assay Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). HiTrapTM Phenyl FF prepacked column was purchased from GE HealthCare (London, UK). The composition of liquid yeast extract peptone dextrose (YPD) medium was 10 g·L−1 yeast extract, 20 g·L−1 tryptone, and 20 g·L−1 glucose. The composition of solid minimal dextrose (MD) medium was 13.4 g·L−1 yeast nitrogen base without amino acids (YNB), 20 g·L−1 glucose, 400 μg·L−1 D-biotin and 15 g·L−1 agar. All other chemicals were analytical or chromatographic grade and were purchased from China National Pharmaceutical Group Corporation (Shanghai, China).

    • The crystal structure of Bos taurus CPA (PDB ID:1M4L) was derived from the PDB database (www.rcsb.org). The three-dimensional structures of mutants were obtained through SWISS-MODEL online server using CPA crystal structures as templates. The quality of 3D models of mutants was evaluated by ERRAT and PROCHECK procedures in SAVES v6.0 online server (https://saves.mbi.ucla.edu/). The structures above the lowest overall quality factor of 90 in REEAT and more than 95% allowed area in Ramachandran plots were used for further analysis[22]. B-Factors of CPA were predicted by HotSpot Wizard 3.0 online procedure (https://loschmidt.chemi.muni.cz/hotspotwizard/). Potential residue pairs for introducing disulfide bonds were synthetically analyzed through Disulfide by Design 2.0 online server (http://cptweb.cpt.wayne.edu/DbD2/index.php) and MODIP online procedure (http://caps.ncbs.res.in/dsdbase2). The position-specific scoring matrix (PSSM) of CPA was generated by the PSI-BLAST algorithm. MD simulations were performed by the Gromacs software following a previously described method by Ming et al. with appropriate modifications[21]. Firstly, it was conducted by Amber99sb-Idn force filed at 370 K for 20 ns. Then, the 3D model of WT and its mutants was wrapped with a water molecule box, respectively. Then, Na+ ions were added to neutralize the charges of the system. The energy minimization using the steepest descent method was subjected to reduce the number of modeled non-normal conformations in the simulated system. Furthermore, a 500-ps isothermal isotropic ensemble (NVT) and isothermal isobaric ensemble (NPT) were executed to balance the entire system, respectively. The RMSF values of WT were calculated to determine the flexible regions of CPA, where residues with RMSF higher than 0.145 nm were considered highly flexible. The Root-Mean-Square Deviation (RMSD) and the root-mean-square fluctuation (RMSF) values of WT and its mutants were determined to assess changes in CPA conformation.

    • The gene sequence of CPA and its mutants (D93C/F96C and K153C/S251C) were optimized based on the codon preference of P. pastoris. Subsequently, they were inserted into the EcoR I and Not I restriction sites of pPIC9K plasmids, respectively. The pPIC9K plasmids containing the cpa gene were stored in E. coli DH5α strains. The above process was constructed by Sangon Biotech Co., Ltd. (Shanghai, China). These recombinant plasmids linearized by Sac I were transferred into P. pastoris competent cells by electrotransformation. The products were successively cultured at 28 °C in histidine-deficient solid minimal dextrose (MD) plates for screening His+ clones. Then, recombinants with different copy numbers of genes were screened by solid YPD mediums containing geneticin G418 at a final concentration of 0.5, 1.0, 2.0, and 4.0 mg·mL−1.

    • The positive clones of CPA and its mutants were cultured at 28 °C, 200 rpm in 25 mL liquid BMGY medium until the OD600 reached 2~6. After the centrifugation at 4,000 rpm for 5 min, the cells were transferred in 20 mL liquid BMGY medium and cultured at 28 °C, 250 rpm for 6 d. The methanol was added to a final concentration of 1% (v/v) every 24 h to induce protein expression. After the centrifugation at 10,000 rpm for 5 min, crude supernatants were analyzed by SDS-PAGE and purified by hydrophobic interaction chromatography. The HiTrapTM Phenyl FF prepacked column was pre-equilibrated in 2 M (NH4)2SO4 (pH 7.4). After fully loaded, the enzymes treated with 2 M (NH4)2SO4 were eluted with 20 mM Tris-HCl (pH 7.4). The SDS-PAGE and Western-Blot analysis of pure enzymes were performed following a previous method described by Ming et al.[21]. And, the concentration of pure enzyme was measured by the Bradford method while using bovine serum albumin (BSA) as the standard.

    • The determination of OTA degradation activity of recombinant CPA and its mutants were detected by HPLC, which was described previously[23]. The activity of recombinant CPA and its mutants was determined using Z-Phe-Leu as the substrate. One unit (U) of activity was defined as the amount of enzyme required to degrade 1 μM Z-Phe-Leu per minute at 37 °C. The reaction system contained 108 μL of Z-Phe-Leu (1 μM ) and 12 μL of purified CPA (10 mg·mL−1), and incubated at 37 °C for 15 min. Then, 240 μL of cadmium-ninhydrin solution was added to it and treated at 85 °C for 5 min. The absorbance of samples at 507 nm was recorded in a spectrophotometer.

    • To determine the optimum temperature (Topt) of recombinant CPA and its mutants, the reaction system contained Z-Phe-Leu, and purified enzymes were incubated at various temperatures ranging from 30 to 70 °C with 10 °C intervals for 15 min. The residual activity was assayed at 30 °C, and the activity at the optimum temperature was considered as 100%.

      To determine the half-time (t1/2) of recombinant CPA and its mutants, the purified enzymes were heat-treated at 65 °C for 10, 20, 30, 40, and 50 min, and then cooled on ice for 10 min. The residual activity was assayed at 30 °C with the initial activity set at 100%. The average thermal inactivation rate constants (Kd) were obtained from the plots of ln (residual activity) vs the incubation time. The value of t1/2 is defined as the time when the residual activity is reduced to half, and it can be calculated by the equation: t1/2 =(ln2/Kd).

      To determine the half-inactivation temperature (T5015) of recombinant CPA and its mutants, the purified enzymes were incubated at temperatures ranging from 40 to 70 °C with 10 °C intervals for 15 min, and then cooled on ice for 10 min. The residual activity was assayed at 30 °C with the initial activity set at 100%. The value of T5015 is the temperature at which the residual activity is decreased to 50% after a 15-min treatment, and it can be generated from the plot of residual activity versus the incubation temperature.

    • The kinetic parameters of purified CPA and its mutants were determined based on the enzyme activity assays. Different final concentrations of Z-Phe-Leu (0.5~2.5 μM) were incubated with CPA at 37 °C for 15 min, respectively. Then, the kinetic parameters of Km and Kcat/Km were calculated by the Line Lineweaver–Burk plots in Origin 2021 software (Origin Lab, USA).

    • The circular dichroism (CD) spectra were conducted on a Pistar II-180 circular dichroism spectrometer (Applied Photophysics, USK). The purified CPA and its mutants prepared in 20 mg·mL−1 Tris−HCl buffer (pH 7.4) were added to the length quartz cuvette with a path length of 0.1 cm. The wavelength range was set from 200 to 260 nm, and the scan speed was 10 nm·s−1. Each scan was repeated three times. The proportion of secondary structures (α-helix, β-sheet, β-turns, and coils) were estimated by CDPro online software (www.bmb.colostate.edu/cdpro).

      The intrinsic fluorescence spectra were performed on a Hitachi F-7000 fluorescence spectrometer (Hitachi, Japan) to evaluate the tertiary structure changes. The purified CPA and its mutants prepared in 20 mmol·L−1 Tris−HCl buffer (pH 7.4) were added to the length quartz cuvette with a path length of 0.1 cm. The excitation wavelength was at 280 nm, and the emission wavelength was recorded ranging from 290 to 500 nm.

    • The disulfide bonds of recombinant CPA and its mutants were quantified mainly according to the content of the free sulfhydryl group when in reduction. Firstly, reduced enzymes were prepared in the following way. They were firstly dissolved in 2 mL disulfide bond reduction buffer and treated at 37 °C for 2 h. Subsequently, 50 μL of TCEP was added to the solution and then treated at 37 °C for 1 h. The solution was to added 2 mL 12% trichloroacetic acid (TCA) and treated at 37 °C for 1 h. After centrifugation at 14,000 g for 10 min, the precipitate was dissolved with 2 mL Tris-Gly buffer. Non-reduced recombinant CPA and its mutants were dissolved in 2 mL Tris-Gly buffer. Fifteen μL of DNTB (5,5'-disulfide (2-nitrobenzene) was added to 1 mL reduced and non-reduced recombinant enzymes, respectively. After the reaction solution was placed at 37 °C for 20 min, the wavelength of 412 nm was detected by ultraviolet spectrophotometer. Disulfide bonds were calculated by the equation: n = N × (A1 − A2)/2 × A1. n was the number of disulfide bonds, N was the number of cysteine residues, A1 was the absorption value of reduced recombination CPA at 412 nm, A2 was the absorption value at 412 nm for non-reduced recombination CPA.

      The ProteinTools online server (https://proteintools.uni-bayreuth.de) was employed to analyze changes in hydrogen bonds between WT and its mutants. PyMOL v2.5 was conducted to visualize alterations in surface charge.

    • All results were expressed as mean value ± standard deviation based on three independent experiments. And, figures were drawn using Origin 2021 software.

    • The enzyme flexible regions with high conformational flexibility are always used as a potential target for molecular modification in protein engineering. The flexible regions of CPA were mainly targeted by two methods (B-factor and RMSF). The B-factor is used to characterize the describe the attenuation of X-ray or neutron scattering caused by thermal motion. Where the higher the B-Factor value, the more flexible the enzyme conformation is[24]. As seen in Fig. 1, the average B-Factor value of residue was calculated by HotSpot Wizard 3.0, and a total of 16 highly flexible regions of CPA were identified.

      Figure 1. 

      Average B-factor values of carboxypeptidase A. Flexible residues were located above the dotted line.

      RMSF represents the amplitude of fluctuation of an atom relative to its average position in the simulated system, reflecting the local flexibility of protein conformation at the atomic level[21]. The higher the RMSF value, the more flexible the enzyme conformation is. As shown in Fig. 2, the RMSF values of CPA were calculated by molecular dynamics simulation, and a total of twelve flexible regions of CPA were screened.

      Figure 2. 

      The RMSF values of carboxypeptidase A. Flexible residues were located above the dotted line.

      To improve the prediction accuracy, B-factor and RMSF values were comprehensively analyzed. Finally, the regions of Ala1-Arg2, Asn5-Thr6, Gly55-Asn58, Gln92-Ser95, Val132-Leu137, Lys153-Ala154, Lys168-Tyr169, Lys231-Tyr234, Try248-Gln249, and Asn306-Asn307 were considered highly flexible regions, laying the foundation for the subsequent rigidification of flexible regions.

    • The introduction of disulfide bonds is always one of the strategies to stabilize the flexible regions. Potential residue pairs for introducing disulfide bonds in CPA were analyzed by two online programs, DbD and MODIP (Supplemental Fig. S1). There were 37 pairs of potential disulfide bonds in CPA predicted by DbD software. Besides, there were eight pairs of potential disulfide bonds predicted by the MODIP program, and they were categorized into Grade A, Grade B, Grade C and Grade D according to the likelihood of disulfide bond formation. To improve the prediction reliability of potential disulfide bonds in CPA, twenty potential disulfide bonds belonging to both DbD and MODIP classes A and B were screened. The residues including Asp93, Ser136 and Lys153 were located in these flexible regions. Therefore, three pairs of residues for introducing disulfide bonds Asp93-Phe96, Ser136-Pro160 and Lys153-Ser251 were initially screened for subsequent analysis.

    • Enzymes not only need enough rigidity to maintain their stability, but also require adequate conformational flexibility to exert its catalytic activity. Thus, there is a trade-off between activity and stability, implying that an increase in stability is accompanied by a concomitant decrease in activity[25]. It was reported that residues associated with the catalytic function of enzymes were typically highly conserved[26]. A total of 151 conserved residues in the CPA sequence were determined by conservativeness analysis of PSSM, containing the screened residue Ser136 (Supplemental Fig. S2). Thus, the disulfide bond Ser136-Pro160 was excluded to prevent the catalytic activity of CPA from being adversely affected when engineering thermostability. Finally, the two CPA disulfide bonds Asp93-Phe96 and Lys153-Ser251 were retained and sequentially named as mutants D93C/F96C and K153C/S251C.

    • Using the crystal structures of CPA (PDB ID:1M4L) as a template, three-dimensional structures of mutants D93C/F96C and K153C/S251C were obtained through SWISS-MODEL online server. The quality of the models was evaluated through the SAVES online platform. The results of Ramachandran plots showed that mutants D93C/F96C and K153C/S251C had more than 95% residues in the favorable regions (Supplemental Fig. S3). And, the results of ERRAT indicated that the overall quality factors of D93C/F96C and K153C/S251C were 95.2703 and 95.5932, respectively (Supplemental Fig. S4). Thus, the three-dimensional models of these two mutants were relatively reliable and closer to their real structures.

    • MD simulations were performed to explore the conformational stability of CPA structure. RMSD reflects the average amount of movement of backbone atoms throughout the whole protein structure, which is negatively correlated with the thermostability of the enzyme[21]. The average RMSD values of mutants D93C/F96C and K153C/S251C were 0.1386 nm (Fig. 3a) and 0.1136 nm (Fig. 3b), which were lower than that of WT (0.1416 nm). It suggested that mutations could make the overall conformation more rigid, contributing to enhancing the thermostability of CPA[27].

      Figure 3. 

      The comparison of RMSD values between wild-type and mutant carboxypeptidase A. (a) WT and D93C/F96C. (b) WT and K153C/S251C.

      The effect of mutation on the local conformational stability of CPA was explored by RMSF. Compared with WT, the mutants D93C/F96C (Fig. 4a) and K153C/S251C (Fig. 4b) both showed a decrease in RMSF values in mutant regions, showing that the mutations stabilize the local conformation of CPA. Taken together, the two mutants D93C/F96C and K153C/S251C possessed the potential to enhance the thermostability compared with WT. And, they were used for the subsequent experimental analysis of CPA thermostability screening.

      Figure 4. 

      The comparison of RMSF values between wild-type and mutant carboxypeptidase A. (a) WT and D93C/F96C. (b) WT and K153C/S251C. The mutant regions are circled in dotted wireframes.

    • The plasmid pPIC9K contains the histidine dehydrogenase sequence (HIS4). It was shown that a large number of single colonies capable of synthesizing histidine grew on the MD plate (Supplemental Fig. S5). The result showed that the recombinant plasmid pPIC9K had been successfully transferred into host cells.

      Because the plasmid pPIC9K contains the kanamycin resistant gene that allows P. pastoris to tolerate G418 disulfate salt. The result showed that the number of viable transformants gradually decreased as the concentration of genistein increased (Supplemental Fig. S6). To some extent, the higher the level of resistance to the genotoxin G418, the higher the copy number of the target gene, which may increase the level of enzyme expression. Therefore, the transformants growing on YPD plates with 4 mg·mL−1 G418 were selected for expression.

      From the SDS-PAGE result of fermentation supernatants, there was a band appeared near 35 kDa, which was more similar to the theoretical molecular weight of CPA (Fig. 5a). The result preliminarily suggested that P. pastoris GS115 realized the heterologous expression of CPA and its mutants. To improve the purity of recombinant enzymes, hydrophobic interaction chromatography was used for purification. And, the SDS-PAGE of purified WT, D93C/F96C and K153C/S251C was shown in Fig. 5b. The purity of purified enzymes exceeded 90% detected by Image J. There were six histidine tags at the N-terminal of recombinant enzymes, the specific bands in the western-blot experiment once again proved that the purified enzymes with a molecular weight of 35 kDa was recombinant CPA and its mutants (Fig. 5c). The concentration of WT, D93C/F96C and K153C/S251 measured by Bradford's method were 0.321, 0.303, and 0.289 mg·mL−1, respectively.

      Figure 5. 

      The SDS-PAGE analysis of (a) fermentation supernatants and (b) purified components expressed by P. pastoris. (c) The western blot analysis of purified components expressed by P. pastoris. M, protein marker (10−180 kDa). 1, WT. 2, D93C/F96C. 3, K153C/S251C. Each sample was prepared by boiling for 5 min and loaded at 20 μL per lane.

    • To determine the OTA degradation efficiency of recombinant CPA and its mutants, the residual OTA in the reaction system was detected by HPLC. It can be seen in Fig. 6 that the peak time of the OTA standard was 4.150 min, and the peak time of the OTα standard was 3.205 min. Compared with the control, the peak area of samples treated with recombinant enzymes decreased significantly at 4.1 min, indicating that OTA was successfully degraded. A new chromatographic peak appeared around 3.2 min, and its retention time was consistent with that of the OTα standard. The result indicated that the OTA degradation product of WT, D93C/F96C and K153C/S251C was OTα.

      Figure 6. 

      HPLC of OTA degradation by recombinant CPA and its mutants.

      It was found that CPA hydrolyzed the amide bond of OTA to generate OTα and L-phenylalanine[21], which was consistent with the principle of hydrolyzing Z-Phe-Leu. Given the toxicity of OTA, Z-Phe-leu was used as a substrate to study the enzymatic properties of CPA. The specific enzyme activity of recombinant WT, D93C/F96C and K153C/S251C was determined by the standard curve of L-leucine solution y = 0.0055x + 0.0857 (R2 = 0.995). The specific enzyme activity was 11.113 ± 0.298, 13.816 ± 0.511, and 10.107 ± 0.255 U·mg−1 for WT, D93C/F96C and K153C/S251C, respectively. The specific enzyme activity of D93C/F96C was increased by 24.32% compared with that of WT, indicating that the introduction of the disulfide bond at sites 93 and 96 did not adversely affect the activity. While the specific enzyme activity of the mutant K153C/S251C was reduced by 9.05% compared with that of WT, showing that the mutations at sites 153 and 251 adversely affected the enzymatic activity of CPA.

    • The reaction system of recombinant enzymes and substrate Z-Phe-Leu was processed at different temperatures for a certain period. And, the residual enzyme activity of recombinant enzymes was measured. As shown in Fig. 7a, the activity increased and then decreased with the increase of reaction temperature, and reached the highest at the optimal temperature. The maximum activity of WT at 40 °C indicated that the optimum temperature of WT was 40 °C. While the optimal temperatures of mutants D93C/F96C and K153/S251 were 10 °C higher than that of WT. The results showed that mutations improved the optimal temperature, which was conducive to the better catalytic activity of CPA at higher temperatures.

      Figure 7. 

      Thermal stability of wild-type and mutant carboxypeptidase A. (a) The optimum temperature of wild-type and mutant carboxypeptidase A. (b) The half-life of wild-type and mutant carboxypeptidase A. (c) Half inactivation temperature of wild-type and mutant carboxypeptidase A.

      The half-life (t1/2) represents the time required for the enzyme to lose half of its activity. The WT, D93C/F96C and K153C/S251C was subjected to treatment at 65 °C for different times, respectively. As shown in Fig. 7b, the half-life curve of WT, D93C/F96C and K153C/S251C was y = −0.05913x + 3.4361 (R2 = 0.98), y = −0.04598x + 3.6209 (R2 = 0.99) and y = 0.04898x + 3.3817 (R2 = 0.97), respectively. Since the slope of the half-life curve is the inactivation rate constant kd, the t1/2 value of WT at 65 °C can be calculated as 11.7 min. The t1/2 of D93C/F96C and K153C/S251C was 15.1 and 14.2 min, respectively, which were 3.4 and 2.5 min higher than that of WT. The above results showed that the heat resistance of CPA was enhanced after the introduction of the disulfide bond, and the activity could be maintained for a longer period at a higher temperature.

      The half-inactivation temperature (T50) refers to the temperature at which the activity drops to 50% of the initial activity. In this study, the recombinant WT, D93C/F96C and K153C/S251C was first subjected to different temperatures for a certain period, respectively. From the half-inactivation temperature curves (Fig. 7c), it can be seen that the activity of WT and its mutants decreased with increasing temperature, but the activity of WT decreased more significantly. After 15 min of treatment at 70 °C, the activity of WT was only 35% of the initial activity, whereas the mutants D93C/F96C and K153C/S251C were able to maintain about 50% of the initial activity. The T5015 of WT was calculated to be 58.0 °C, while the T5015 of mutants D93C/F96C and K153C/S251C were 66.5 and 69.4 °C, which were 8.5 and 11.4 °C higher than that of WT, respectively. The above results indicated that mutants D93C/F96C and K153C/S251C were more stable and could maintain better activity at higher temperatures.

    • The enzyme kinetic constants, Km and Kcat/Km, were measured to evaluate the enzymatic properties of WT and its mutants. The Michaelis constant Km reflects the affinity of the enzyme for the substrate. The smaller the value is, the better the affinity of the enzyme for the substrate. As shown in Table 1, the Km of D93C/F96C and K153C/S251 were not significantly different from those of WT, indicating that the mutation did not affect the affinity for the substrate. The Kcat/Km denotes the catalytic efficiency of the enzyme. And, the larger the value is, the higher the catalytic efficiency of the enzyme is. As shown in Table 1, the Kcat/Km of D93C/F96C was the largest, whose catalytic efficiency was increased by 43.15% compared with that of WT. While, the Kcat/Km value of K153C/S251 was decreased by 8.08%, demonstrating that its catalytic efficiency was slightly lower than that of WT.

      Table 1.  Kinetic parameters of wild-type and mutant carboxypeptidase A.

      Enzyme Km (μM) Vmax (μM·min−1) Kcat/Km (μM−1·s−1)
      WT 0.277 ± 0.012 1.833 ± 0.014 4.549 × 10−3
      D93C/F96C 0.271 ± 0.021 2.569 ± 0.036 6.512 × 10−3
      K153C/S251C 0.268 ± 0.043 1.641 ± 0.047 4.209 × 10−3

      The above results of recombinant WT, D93C/F96C and K153C/S251C were similar to those of specific enzyme activities. It indicated that D93C/F96C achieved a dual improvement in thermostability and activity. Zhou et al. also found that the stability and activity of LPMOs could be improved simultaneously[11]. While the catalytic activity of K153C/S251C suffered from undesired effects when engineering thermostability, exhibiting a prevalent phenomenon 'stability-activity trade-off'[25]. Ming et al. also reported a similar result that the OTA degradation ability of CPA mutant R124K and S134P decreased to varying degrees while improving their thermostability[21].

    • The circular dichroic absorption spectra of recombinant CPA at 200~260 nm were scanned to investigate the effect of mutations on CPA secondary structure. Subsequently, the percentage content of secondary structures such as α-helixes, β-strands, β-turns and coils were determined by CDpro computer software. As shown in Fig. 8, the contents of α-helixes, β-strands, β-turns, and coils for WT were 24.6%, 26.6%, 21.6%, and 27.5%, respectively. Compared with WT, the mutants D93C/F96C and K153C/S251C showed an increase in α-helixes by 5.1% and 10.7%, while a decrease in β-strands, β-turns and coils. The above results indicated that the increase of α-helixes might be a key cause of their structural preservation[19], which was conducive to the improvement of the thermostability of CPA.

      Figure 8. 

      The contents of secondary structures (α-helix, β-strand, β-turn, and coil) in CPA and its mutants.

      The fluorescence emission spectra of recombinant CPA at 290~500 nm was scanned to investigate the effect of mutation on CPA tertiary structure. As shown in Fig. 9, WT and its mutants D93C/F96C and K153C/S251C all had a maximum emission at around 335 nm, demonstrating the characteristics of tryptophan fluorescence[28]. It suggested that there was no obvious impact on the tertiary structure of CPA when introducing disulfide bonds. A similar result was reported in 1,4-α-glucan branching enzyme. Introducing disulfide bonds might only effect the secondary structure of it while the tertiary structure continued a similar trend[29].

      Figure 9. 

      Intrinsic fluorescence spectra of wild-type and mutant carboxypeptidase A.

    • The formation of disulfide bonds was quantified by detecting the amount of free sulfhydryl groups by DNTB[30]. As shown in Table 2, it was determined that WT possessed no natural disulfide bond. While, the mutants D93C/F96C and K153C/S251C both had one disulfide bond. The above results indicated that the mutants D93C/F96C and K153C/S251C successfully formed intramolecular disulfide bonds, realizing the purpose of enhancing the thermostability of CPA by introducing disulfide bonds.

      Table 2.  Comparison of disulfide bond number between wild-type and mutant carboxypeptidase A.

      Enzyme A1 A2 Disulfide bond
      WT 0.456 ± 0.009 0.342 ± 0.010 0
      D93C/F96C 1.434 ± 0.035 0.367 ± 0.018 1
      K153C/S251C 1.549 ± 0.050 0.464 ± 0.023 1

      The mutation of residues often leads to complex changes in multiple intramolecular interactions. In addition to analyzing disulfide bonds, ProteinTools online software was conducted to investigate the changes in the number of hydrogen bonds in mutant regions. Compared with WT, the mutant D93C/F96C showed an increase from one to two hydrogen bonds at mutation sites 93 and 96, increasing the thermostability by maintaining the rigidity of the enzyme structure (Fig. 10a). While, the mutant K153C/S251C lost the hydrogen bond formed between the mutation site 153 and 251, which might result in the decrease of enzyme activity (Fig. 10b). This was because hydrogen bonds were important factors for stabilizing the enzyme secondary structure[6].

      Figure 10. 

      Comparison of the number of hydrogen bonds in mutant regions between wild-type and its mutant carboxypeptidase A. (a) WT and D93C/F96C. (b) WT and K153C/S251C.

      The changes in surface charges for WT and its mutants were analyzed by PyMOL software. The enhanced thermostability of D93C/F96C and K153C/S251C might also be related to the surface charge redistribution in mutant regions. As shown in Fig. 11a, the WT was electrically neutral at the sites of Asp93 and Phe96, with the region of Asp93-Phe96 showing electronegative. Whereas residues at sites 93 and 96 were both mutated to Cys, the mutant D93C/F96C exhibited positive electronegativity at sites 93 and 96. And, the mutant region shifted from negative to positive electronegativity. The phenomenon might be caused by the reduction of a carboxyl group and the formation of two sulfhydryl groups in residue structures. For mutant K153C/S251C, when replacing positively charged Lys at site 153 and positively charged Ser at site 251 with uncharged Cys, not only did the surface charges of mutation sites change from positive to neutral, but also the surface charges of the mutation region Lys153-Ser251 showed a similar trend (Fig. 11b). The disappearance of an amino group and a hydroxyl group and the formation of two sulfhydryl groups could be associated with the changes in surface charge of K153C/S251C. Chen et al. also reported a similar result that the redistribution of surface electrostatic charges enhanced the thermostability of glycosyltransferase UGT76G1[30]. Besides, Arabnejad et al. found that the positive effect on thermostability of halohydrin dehalogenase D162T could be contributed to the redistribution of surface electrostatic charges caused by the removal of the carboxyl group[31].

      Figure 11. 

      Comparison of surface charge distribution in mutant regions between wild-type and mutant carboxypeptidase A. (a) WT and D93C/F96C; (b) WT and K153C/S251C. The mutation regions were circled in black wireframes.

    • A rational design of disulfide bonds was employed to enhance CPA thermostability. Two CPA mutants, D93C/F96C and K153C/S251C, were successfully designed and experimentally proved to possess enhanced thermostability. The mechanism of enhanced thermostability was related to changes in secondary structure and intramolecular interactions. This study showed that the rational design of disulfide bonds was an effective strategy to enhance the thermostability of CPA, which was helpful in broadening the applicability of CPA in industrial fields such as OTA degradation, bitter taste removal and so on.

    • The authors confirm contribution to the paper as follows: study conception and design: Zhang H, Liang Z; data collection: Zhang H, Zhao Z; analysis and interpretation of results: Zhang H, Zhao Z, Liang Z; draft manuscript preparation: Zhang H, Liang Z. All authors reviewed the results and approved the final version of the manuscript.

    • All data generated or analyzed during this study are included in this published article.

      • This research was funded by the National Key Research and Development Program of China (No. 2023YFD1301000); and the Shandong Province Natural Science Foundation (No. ZR202102260301).

      • The authors declare that they have no conflict of interest.

      • Supplemental Fig. S1 The residue pairs forming potential disulfide bridges of carboxypeptidase A.
      • Supplemental Fig. S2 The Sequence conservation of carboxypeptidase A.
      • Supplemental Fig. S3 The ERRAT evaluation of mutant carboxypeptidase A D93C/F96C (a) and K153C/S251C (b).
      • Supplemental Fig. S4 The Ramachandran plots of mutant carboxypeptidase A D93C/F96C (a) and K153C/S251C (b).
      • Supplemental Fig. S5 Screening His+ transformations by MD plates of WT (a), D93C/F96C (b) and K153C/S251C (c).
      • Supplemental Fig. S6 Screening of high-copy strains by G418 with gradient concentrations. (a) WT. (b)D93C/F96C. (c) K153C/S251C.
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of China Agricultural University, Zhejiang University and Shenyang Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (11)  Table (2) References (31)
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    Zhang H, Zhao Z, Zhu M, Logrieco AF, Wang H, et al. 2024. Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds. Food Innovation and Advances 3(2): 191−201 doi: 10.48130/fia-0024-0017
    Zhang H, Zhao Z, Zhu M, Logrieco AF, Wang H, et al. 2024. Enhancing the thermostability of carboxypeptidase A by rational design of disulfide bonds. Food Innovation and Advances 3(2): 191−201 doi: 10.48130/fia-0024-0017

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