Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

ABSTRACT In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (k cat/Km ) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

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Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Applied and Environmental Microbiology ◽

10.1128/aem.07012-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3880-3884 ◽

Cited By ~ 17

Author(s):

Yu-Ri Lim ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Content Type ◽

Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1600426x ◽

2016 ◽

Vol 72 (4) ◽

pp. 328-335 ◽

Cited By ~ 10

Author(s):

Zhongchuan Liu ◽

Tian Xie ◽

Qiuping Zhong ◽

Ganggang Wang

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

The Novel ◽

Wild Type ◽

Outer Coat ◽

Cota Laccase ◽

Key Residues

The CotA laccase fromBacillus subtilisis an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

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Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities

Journal of Bacteriology ◽

10.1128/jb.187.21.7543-7545.2005 ◽

2005 ◽

Vol 187 (21) ◽

pp. 7543-7545 ◽

Cited By ~ 3

Author(s):

Chew Ling Tan ◽

Chew Chieng Yeo ◽

Hoon Eng Khoo ◽

Chit Laa Poh

Keyword(s):

Catalytic Efficiency ◽

Relative Activity ◽

Site Directed Mutagenesis ◽

Mutant Enzyme ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Specific Mutation ◽

Catalytic Activities ◽

Pseudomonas Alcaligenes

ABSTRACT xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k cat/Km ) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

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Site-Directed Mutagenesis of a Neutral Phytase from Bacillus amyloliquefaciens: Influencing Activity and Stability

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.1033-1034.271 ◽

2014 ◽

Vol 1033-1034 ◽

pp. 271-278 ◽

Cited By ~ 2

Author(s):

Wei Xu ◽

Zu Peng Wang ◽

Rong Shao

Keyword(s):

Bacillus Amyloliquefaciens ◽

Specific Activity ◽

Fluorescence Emission ◽

Emission Spectra ◽

Feed Additives ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Sequence Alignments ◽

Fluorescence Emission Spectra

In order to improve the activity and stability of phytase fromBacillus amyloliquefaciens, site-directed mutagenesis has been performed base on the previous recombinantE.coliBL21 harboring the expression vector ofphyC. Mutation residues were chosen based on the sequence alignments and structure analysis of neutral phytsaes from different microorganisms. Site-directed mutagenesis techniques were used to get three mutants (D148E/H149R, Q67E/N68R, and D191E), then the mutants were expressed and purified. Enzymatic characters of different mutants were investigated. The results indicated that the optimum pH of all mutants were 7.0, and the optimum temperature were between 65 °C–70 °C. The maximum specific activity of mutant D148E/H149E was 27.84 U/mg which was 2.19 times than that of the wild-type phytase. The half inactivation temperature of D191E was 4.5 °C higher than that of the wild-type phytase. Fluorescence emission spectra showed that slight differences were among the structures of the mutant phytases. The phytases described here which have increased activity and thermostability may have promosing potential as feed additives in animal diets.

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The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

Biochemical Journal ◽

10.1042/bj2950485 ◽

1993 ◽

Vol 295 (2) ◽

pp. 485-491 ◽

Cited By ~ 4

Author(s):

G Zapata ◽

P P Roller ◽

J Crowley ◽

W F Vann

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Wild Type ◽

Acetylneuraminic Acid ◽

Denatured States ◽

Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

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Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation

Biochemical Journal ◽

10.1042/bj3010275 ◽

1994 ◽

Vol 301 (1) ◽

pp. 275-281 ◽

Cited By ~ 35

Author(s):

H M Chen ◽

C Ford ◽

P J Reilly

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Membrane ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Aspergillus Awamori ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Recognition Sites ◽

Membrane Components

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

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Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

Biochemical Journal ◽

10.1042/bj3260047 ◽

1997 ◽

Vol 326 (1) ◽

pp. 47-51 ◽

Cited By ~ 16

Author(s):

Shoshana KEYNAN ◽

Nigel M. HOOPER ◽

Anthony J. TURNER

Keyword(s):

Specific Activity ◽

Mammalian Species ◽

Competitive Inhibitor ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Renal Metabolism ◽

Leukotriene D4 ◽

Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

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Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3123-3126 ◽

Cited By ~ 5

Author(s):

Carlo Bottoni ◽

Mariagrazia Perilli ◽

Francesca Marcoccia ◽

Alessandra Piccirilli ◽

Cristina Pellegrini ◽

...

Keyword(s):

Catalytic Activity ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Zinc Ion ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

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Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00457-13 ◽

2013 ◽

Vol 79 (13) ◽

pp. 4072-4077 ◽

Cited By ~ 57

Author(s):

Xuguo Duan ◽

Jian Chen ◽

Jing Wu

Keyword(s):

Catalytic Efficiency ◽

Kinetic Studies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Ph Optimum ◽

Widespread Application ◽

Content Type ◽

Major Factors

ABSTRACTPullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase fromBacillus deramificanswere selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that theKmvalues for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and theKcat/Kmvalues increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease

Frontiers in Chemistry ◽

10.3389/fchem.2021.692168 ◽

2021 ◽

Vol 9 ◽

Author(s):

Juliana C. Ferreira ◽

Samar Fadl ◽

Adrian J. Villanueva ◽

Wael M. Rabeh

Keyword(s):

Catalytic Activity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Oligomeric State ◽

Wild Type Enzyme ◽

Complete Inactivation ◽

Catalytic Function ◽

Catalytic Inactivation ◽

Catalytic Dyad

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

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