Codon optimality is the primary contributor to the exceptional mutational sensitivity of CcdA antitoxin in its operonic context

Deep mutational scanning studies suggest that single synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin operonic system is unusually sensitive to mutations when studied in the operonic context. A large fraction (~80%) of single codon mutations, including many synonymous mutations in the ccdA gene show inactive phenotypes that are correlated with the E.coli codon usage frequency but retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure or stability, consistent with the fact that a large region of CcdA is intrinsically disordered. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that these mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by predicting and validating single synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their native context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred from previous saturation mutagenesis studies.

A Sustainable Approach for Synthesizing (R)-4-Aminopentanoic Acid From Levulinic Acid Catalyzed by Structure-Guided Tailored Glutamate Dehydrogenase

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.

A Saturation-Mutagenesis Analysis of the Interplay Between Stability and Activation in Ras

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H?Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H?Ras in mammalian Ba/F3 cells correlate well with results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase?activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g., at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen?deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with the destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli

Chiral amino alcohols are prevalent synthons in pharmaceuticals and synthetic bioactive compounds. The efficient synthesis of chiral amino alcohols using ammonia as the sole amino donor under mild conditions is highly desired and challenging in organic chemistry and biotechnology. Our previous work explored a panel of engineered amine dehydrogenases (AmDHs) derived from amino acid dehydrogenase (AADH), enabling the one-step synthesis of chiral amino alcohols via the asymmetric reductive amination of α-hydroxy ketones. Although the AmDH-directed asymmetric reduction is in a high stereoselective manner, the activity is yet fully excavated. Herein, an engineered AmDH derived from a leucine dehydrogenase from Sporosarcina psychrophila (SpAmDH) was recruited as the starting enzyme, and the combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to improve the activity. After three rounds of mutagenesis in an iterative fashion, the best variant wh84 was obtained and proved to be effective in the asymmetric reductive amination of 1-hydroxy-2-butanone with 4-fold improvements in kcat/Km and total turnover number (TTN) values compared to those of the starting enzyme, while maintaining high enantioselectivity (ee >99%) and thermostability (T5015 >53°C). In preparative-scale reaction, the conversion of 100 and 200 mM 1-hydroxy-2-butanone catalyzed by wh84 was up to 91–99%. Insights into the source of an enhanced activity were gained by the computational analysis. Our work expands the catalytic repertoire and toolbox of AmDHs.

Missense variants causing Wiedemann-Steiner syndrome preferentially occur in the KMT2A-CXXC domain and are accurately classified using AlphaFold2

Wiedemann-Steiner syndrome (WSS) is a neurodevelopmental disorder caused by de novo variants in KMT2A, which encodes a multi–domain histone methyltransferase. To gain insight into the currently unknown pathogenesis of WSS, we examined the spatial distribution of likely WSS–causing variants across the 15 different domains of KMT2A. Compared to variants in healthy controls, WSS variants exhibit a 64.1–fold overrepresentation within the CXXC domain – which mediates binding to unmethylated CpGs – suggesting a major role for this domain in mediating the phenotype. In contrast, we find no significant overrepresentation within the catalytic SET domain. Corroborating these results, we find that hippocampal neurons from Kmt2a–deficient mice demonstrate disrupted H3K4me1 preferentially at CpG-rich regions, but this has no systematic impact on gene expression. Motivated by these results, we combine accurate prediction of the CXXC domain structure by AlphaFold2 with prior biological knowledge to develop a classification scheme for missense variants in the CXXC domain. Our classifier achieved 96.0% positive and 92.3% negative predictive value on a hold–out test set. This classification performance enabled us to subsequently perform an in silico saturation mutagenesis and classify a total of 445 variants according to their functional effects. Our results yield a novel insight into the mechanistic basis of WSS and provide an example of how AlphaFold2 can contribute to the in silico characterization of variant effects with very high accuracy, establishing a paradigm potentially applicable to many other Mendelian disorders.

Computational Saturation Mutagenesis of SARS-CoV-1 Spike Glycoprotein: Stability, Binding Affinity, and Comparison With SARS-CoV-2

Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we per-formed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003–2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003–2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the S glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.

Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability

The chitosanase (BaCsn46A) was extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in E. coli BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60°C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

Background Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. Results We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively. Conclusions In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.