The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN′

ABSTRACT To ascertain whether position 131 of a mesophilic protease, subtilisin BPN′, is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492–495, 1998), a full set of subtilisin BPN′ mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN′ and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k cat/Km ) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k cat and a decrease inKm . All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.

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Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽

10.1182/blood.v104.11.1725.1725 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1725-1725 ◽

Cited By ~ 1

Author(s):

Hironao Wakabayashi ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Saturation Mutagenesis ◽

Single Amino Acid ◽

Single Point Mutation ◽

Wild Type ◽

Activity Increase ◽

Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

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Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase

Canadian Journal of Microbiology ◽

10.1139/w01-118 ◽

2001 ◽

Vol 47 (12) ◽

pp. 1088-1094 ◽

Cited By ~ 21

Author(s):

Yew-Loom Chen ◽

Tsung-Yin Tang ◽

Kuo-Joan Cheng

Keyword(s):

Amino Acid ◽

Directed Evolution ◽

Catalytic Domain ◽

Specific Activity ◽

Synergistic Effects ◽

High Specific Activity ◽

Site Directed Mutagenesis ◽

Amino Acid Substitutions ◽

Wild Type ◽

Neocallimastix Patriciarum

The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10 128 µmol glucose·min1·(mg protein)1 at pH 6.0. It was more thermostable at 60°C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.Key words: xylanase, Neocallimastix patriciarum, alkalophilicity, random mutagenesis, directed evolution.

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Overproduction of l-Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3064-3070.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3064-3070 ◽

Cited By ~ 22

Author(s):

Hiroaki Motoyama ◽

Hiroshi Yano ◽

Yoko Terasaki ◽

Hideharu Anazawa

Keyword(s):

Amino Acid ◽

Specific Activity ◽

Test Tube ◽

Amino Acid Sequences ◽

Nutritional Requirement ◽

Wild Type ◽

Gene Encoding ◽

Lysine Production ◽

Obligate Methylotroph ◽

In The Wild

ABSTRACT The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by l-lysine, was cloned from an l-threonine- andl-lysine-coproducing mutant of the obligate methylotrophMethylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapAmutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an l-threonine producing mutant, elevated the specific activity of DDPS 20-fold andl-lysine production 2- to 3-fold with concomitant reduction of l-threonine in test tube cultures. AL119 containing thedapA gene produced 8 g of l-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M r of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition byl-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector,l-lysine.

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Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.01176-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7291-7299 ◽

Cited By ~ 22

Author(s):

Mirella Di Lorenzo ◽

Aurelio Hidalgo ◽

Rafael Molina ◽

Juan A. Hermoso ◽

Domenico Pirozzi ◽

...

Keyword(s):

Staining Method ◽

Rhizopus Oryzae ◽

Specific Activity ◽

Oxidation Products ◽

Saturation Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Wild Type Enzyme ◽

Lipid Oxidation Products ◽

The Stability

ABSTRACT A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.

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WHIRLY1 maintains leaf photosynthetic capacity in tomato by regulating the expression of RbcS1 under chilling stress

Journal of Experimental Botany ◽

10.1093/jxb/eraa145 ◽

2020 ◽

Vol 71 (12) ◽

pp. 3653-3663 ◽

Cited By ~ 3

Author(s):

Kunyang Zhuang ◽

Jieyu Wang ◽

Baozhen Jiao ◽

Chong Chen ◽

Junjie Zhang ◽

...

Keyword(s):

Amino Acid ◽

Low Temperature ◽

Cold Adaptation ◽

Promoter Region ◽

Photosynthetic Capacity ◽

Chilling Stress ◽

Wild Type ◽

Quantitative Real Time Pcr ◽

Rbcs Genes ◽

Chilling Treatment

Abstract Rubisco, which consists of eight large subunits (RBCLs) and eight small subunits (RBCSs), is a major photosynthetic enzyme that is sensitive to chilling stress. However, it is largely unclear how plants maintain high Rubisco content under low temperature conditions. Here, we report that tomato WHIRLY1 (SlWHY1) positively regulates the Rubisco level under chilling stress by directly binding to the promoter region of SlRbcS1, resulting in the activation of SlRbcS1 expression. SlRbcS1-overexpressing lines had higher Rubisco contents and were more resistant to chilling stress compared with the wild type. Quantitative real-time PCR analyses showed that, among the five RbcS genes, only SlRbcS1 expression is up-regulated by chilling treatment. These results indicate that SlWHIRLY1 specifically enhances the levels of SlRbcS1 and confers tolerance to chilling stress. The amino acid sequence of SlRBCS1 shows 92.67% identity with those of another two RBCS proteins and three residues are specifically found in SlRBCS1. However, mutation of these residues to alanine in SlRBCS1 does not influence its function during cold adaptation. Thus, we conclude that high levels of Rubisco, but not the specific residues in SlRBCS1, play important roles in tolerance to chilling stress in tomato.

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The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.1.63-67.2000 ◽

2000 ◽

Vol 44 (1) ◽

pp. 63-67 ◽

Cited By ~ 82

Author(s):

David C. Lamb ◽

Diane E. Kelly ◽

Theodore C. White ◽

Steven L. Kelly

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Amino Acid Substitution ◽

Specific Activity ◽

Difference Spectrum ◽

Microsomal Protein ◽

Ergosterol Biosynthesis ◽

Wild Type ◽

Binding Studies ◽

Directed Mutation

ABSTRACT The cytochrome P450 sterol 14α-demethylase (CYP51) ofCandida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

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Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Frontiers in Catalysis ◽

10.3389/fctls.2021.774512 ◽

2021 ◽

Vol 1 ◽

Author(s):

Eun Young Hong ◽

Sun-Gu Lee ◽

Hyungdon Yun ◽

Byung-Gee Kim

Keyword(s):

Disulfide Bond ◽

Specific Activity ◽

Catalytic Efficiency ◽

Arginine Decarboxylase ◽

Saturation Mutagenesis ◽

Alkaline Ph ◽

Wild Type ◽

Active Site Residues ◽

Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).

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Factor IX Padua: From Biochemistry to Gene Therapy

Blood ◽

10.1182/blood.v128.22.sci-9.sci-9 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-9-SCI-9

Author(s):

Valder Arruda ◽

Ben J. Samelson-Jones

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Amino Acid ◽

Early Phase ◽

Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Amino Acid Substitutions ◽

Wild Type ◽

Active Variant

Abstract Novel approaches to enhance the biologic activity of therapeutic proteins have the potential to improve protein- and gene-based therapy for hemophilia. We have identified the variant Factor IX Padua (FIX) (R338L) with 8-fold increase in specific activity compared to wild-type FIX as well as additional strategies to identify other modifications with a positive effect on the biological activity of FIX. FIX-Padua is already in early phase gene therapy clinical trials for hemophilia B. However, it is notable that the field is moving forward even though the molecular basis for its enhanced function has remained elusive. The increased specific activity of FIX Padua compared to FIX wild-type resides in the activated protease as purified recombinant FIX Padua displays enhanced clotting activity as both a zymogen and activated protease. This augmentation of FIX Padua zymogen and protease is observed in both clotting and thrombin generation assays. However, preliminary biochemical characterization suggests that that the increased activity is most pronounced in plasma-based assays, while differences in enzyme kinetic parameters measured in reconstituted systems are more modest. Intriguingly, we have found that most amino acid substitutions at position 338, result in a FIX variant with comparable or enhanced clotting activity with the Padua substitution resulting in the most active variant, suggesting that R338 in FIX wild-type forms an unfavorable interaction that can be relieved by most amino acid substitutions. The wild-type variant is actually the least active variant at this position that is not known to cause hemophilia B. Since, R338 is strictly conserved among mammalian FIX orthologues, there may be an evolutionary pressure to maintain the unfavorable interactions of R338 and limit FIX activity. The corollary to this speculation is that other FIX mutations that relieve deleterious interactions will also increase clotting activity. The characterization of FIX Padua suggests small biochemical improvements may result in substantial increases in plasma based clotting activity. Promising preclinical studies on efficacy and safety, including thrombogenicity and immunogenicity, in small and large animal models provide the basis for translational studies using these proteins. These studies support the concept that the thrombotic risk of FIX Padua activity is similar to FIX wild-type activity. The immunogenicity of FIX Padua is comparable to FIX wild-type in either an adeno-associated virus-based muscle- or liver-directed gene therapy in canine models of hemophilia B. In the last 18 months, results from first 10 men with severe hemophilia B enrolled in two ongoing AAV liver-directed gene therapy clinical trials using a FIX Padua as a transgene were reported. No subject in either study developed inhibitors to FIX Padua or thrombotic complications. In subjects with sustained FIX Padua expression, FIX activity was greater than 10%. These promising early phase results demonstrate the potential of utilizing variants with increased specific activity in gene therapy allowing for lower therapeutic vector doses. It remains to be seen if curative factor levels can be safely achieved with further vector refinements including improved FIX variants. Disclosures Arruda: Pfizer: Research Funding.

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Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10960-x ◽

2020 ◽

Vol 104 (24) ◽

pp. 10515-10529

Author(s):

Sanni Voutilainen ◽

Markus Heinonen ◽

Martina Andberg ◽

Emmi Jokinen ◽

Hannu Maaheimo ◽

...

Keyword(s):

Machine Learning ◽

Amino Acid ◽

Protein Engineering ◽

Specific Activity ◽

Complex Structure ◽

Feature Learning ◽

Wild Type ◽

Learning Methods ◽

Clear Cut ◽

Machine Learning Methods

Abstract In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. Key points • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.

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Packpred: Predicting the functional effect of missense mutations

10.1101/2020.12.30.424909 ◽

2021 ◽

Author(s):

Kuan Pern Tan ◽

Tejashree Rajaram Kanitkar ◽

Kwoh Chee Keong ◽

M.S. Madhusudhan

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Substitution Matrix ◽

Saturation Mutagenesis ◽

Data Sets ◽

Missense Mutations ◽

Wild Type ◽

Data Set ◽

Functional Consequences

1.AbstractPredicting the functional consequences of single point mutations has relevance to protein function annotation and to clinical analysis/diagnosis. We developed and tested Packpred that makes use of a multi-body clique statistical potential in combination with a depth dependent amino acid substitution matrix (FADHM) and positional Shannon Entropy to predict the functional consequences of point mutations in proteins. Parameters were trained over a saturation mutagenesis data set of T4-lysozyme (1966 mutations). The method was tested over another saturation mutagenesis data set (CcdB; 1534 mutations) and the Missense3D data set (4099 mutations). The performance of Packpred was compared against those of six other contemporary methods. With MCC values of 0.42, 0.47 and 0.36 on the training and testing data sets respectively, Packpred outperforms all method in all data sets, with the exception of marginally underperforming to FADHM in the CcdB data set. On analyzing the results, we could build meta servers that chose best performing methods of wild type amino acids and for wild type-mutant amino acid pairs. This lead to an increase of MCC value of 0.40 and 0.51 for the two meta predictors respectively on the Missense3D data set. We conjecture that it is possible to improve accuracy with better meta predictors as among the 7 methods compared, at the least one method or another is able to correctly predict ∼99% of the data.

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