scholarly journals An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Joanna Griffin ◽  
Paul C. Engel
Keyword(s):  
Positive Charge ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Coenzyme Specificity ◽  
E Coli ◽  
Coenzyme Binding ◽  
The Core ◽  
Clostridium Symbiosum ◽  
Key Residues

Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH). A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH. At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.

scholarly journals Site Saturation Mutagenesis Applications onCandida methylicaFormate Dehydrogenase

Scientifica ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Gülşah P. Özgün ◽  
Emel B. Ordu ◽  
H. Esra Tütüncü ◽  
Emrah Yelboğa ◽  
Richard B. Sessions ◽  
...  
Keyword(s):  
Rational Design ◽  
Catalytic Domain ◽  
Residual Activity ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Nadh Regeneration ◽  
Coenzyme Specificity ◽  
Site Saturation ◽  
Coenzyme Binding ◽  
Conserved Residue

In NADH regeneration,Candida methylicaformate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependentcmFDH from NAD+to NADP+and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+to5.6×104-fold and5×104-fold value, respectively. To increase the thermostability ofcmFDH, the conserved residue at position 1 in the catalytic domain ofcmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties ofcmFDH.

scholarly journals Basic residues play key roles in catalysis and NADP+-specificity in maize (Zea mays L.) photosynthetic NADP+-dependent malic enzyme

2004 ◽  
Vol 382 (3) ◽  
pp. 1025-1030 ◽  
Author(s):  
Enrique DETARSIO ◽  
Carlos S. ANDREO ◽  
María F. DRINCOVICH
Keyword(s):  
Zea Mays ◽  
Malic Enzyme ◽  
Zea Mays L ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Reciprocal Inhibition ◽  
Partition Ratio ◽  
Site Directed Mutagenesis ◽  
Three Dimensional Model ◽  
Coenzyme Specificity

C4-specific (photosynthetic) NADP+-dependent malic enzyme (NADP+-ME) has evolved from C3-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP+-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP+-ME K255I (Lys-255→Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP+-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play ‘back-up’ roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP+ versus NAD+) of maize NADP+-ME by interacting with the 2′-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP+ or NAD+, as well as by the reciprocal inhibition constants of the competitive inhibitors 2′-AMP and 5′-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436→Ile) with wild-type NADP+-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP+-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

scholarly journals The Role of Ala198 in the Stability and Coenzyme Specificity of Bacterial Formate Dehydrogenases

Acta Naturae ◽  
2015 ◽  
Vol 7 (1) ◽  
pp. 60-69 ◽  
Author(s):  
A. A. Alekseeva ◽  
V. V. Fedorchuk ◽  
S. A. Zarubina ◽  
E. G. Sadykhov ◽  
A. D. Matorin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermal Inactivation ◽  
Fold Increase ◽  
Amino Acid Replacement ◽  
Site Directed Mutagenesis ◽  
Inactivation Kinetics ◽  
Pseudomonas Sp ◽  
Coenzyme Specificity ◽  
Coenzyme Binding ◽  
The Stability

It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles and . These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the K M NAD+ value from 60 to 35 and from 80 to 45 M for PseFDH and MorFDH, respectively, while the K M HCOO- value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme specificity changed from NAD + to NADP +. In this case, an increase in thermal stability was also observed, but the influence of the mutation on the kinetic parameters was opposite: KM increased from 190 to 280 M and from 43 to 89 mM for NADP + and formate, respectively. According to the data obtained, inference could be drawn that earlier formate dehydrogenase from bacterium Pseudomonas sp. 101 was specific to NADP +, but not to NAD +.

scholarly journals A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

2016 ◽  
Vol 82 (9) ◽  
pp. 2718-2727 ◽  
Author(s):  
Wei Xia ◽  
Haiqiang Lu ◽  
Mengjuan Xia ◽  
Ying Cui ◽  
Yingguo Bai ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Binding Pockets ◽  
Alicyclobacillus Acidocaldarius ◽  
Key Residues

ABSTRACTFew members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-β-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilicAlicyclobacillussp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpartAlicyclobacillus acidocaldariusManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of β-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support −4 to −1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 β-mannanases and distinguishes them from counterparts of other families.

scholarly journals Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

2012 ◽  
Vol 56 (3) ◽  
pp. 1151-1156 ◽  
Author(s):  
Sandrine Dahyot ◽  
Hedi Mammeri
Keyword(s):  
Hot Spot ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Hydrolysis Rate ◽  
Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Class A ◽  
Extended Spectrum ◽  
Cloning Experiment

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

scholarly journals Engineering Promiscuous Alcohol Dehydrogenase Activity of a Reductive Aminase AspRedAm for Selective Reduction of Biobased Furans

2021 ◽  
Vol 9 ◽  
Author(s):  
Hao-Yu Jia ◽  
Zi-Yue Yang ◽  
Qi Chen ◽  
Min-Hua Zong ◽  
Ning Li
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Selective Reduction ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
E Coli ◽  
Alcohol Dehydrogenase Activity ◽  
Starting Point

Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (kcat/Km, 23.6 mM−1 s−1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM−1 s−1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35–95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).

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

2005 ◽  
Vol 71 (9) ◽  
pp. 5290-5296 ◽  
Author(s):  
Wei Zhang ◽  
Yuan Liu ◽  
Huabao Zheng ◽  
Sheng Yang ◽  
Weihong Jiang
Keyword(s):  
Specific Activity ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Catalytic Function ◽  
Alkaline Shift ◽  
Key Residues

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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