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 +.

Making biochemistry count: life among the amino acid dehydrogenases

2011 ◽  
Vol 39 (2) ◽  
pp. 425-429 ◽  
Author(s):  
Paul C. Engel
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Coenzyme Binding ◽  
Guiding Principle ◽  
Glutamate Binding ◽  
Metabolic Role ◽  
Structure Solution ◽  
Unexpected Outcomes ◽  
Insight Into

The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the ‘wrong’ enantiomer in engineered mutant enzymes and implications for resolution of racemates.

scholarly journals Additivity of the Stabilization Effect of Single Amino Acid Substitutions in Triple Mutants of Recombinant Formate Dehydrogenase from the Soybean Glycine max

Acta Naturae ◽  
2015 ◽  
Vol 7 (3) ◽  
pp. 55-64 ◽  
Author(s):  
A. A. Alekseeva ◽  
I. S. Kargov ◽  
S. Yu. Kleimenov ◽  
S. S. Savin ◽  
V I. Tishkov
Keyword(s):  
Thermal Stability ◽  
Glycine Max ◽  
Amino Acid ◽  
Formate Dehydrogenase ◽  
Single Amino Acid ◽  
Inactivation Kinetics ◽  
Amino Acid Substitutions ◽  
Stabilization Effect ◽  
The Stability ◽  
Thermal Stability Of

Recently, we demonstrated that the amino acid substitutions Ala267Met and Ala267Met/Ile272Val (Alekseeva et al., Biochemistry, 2012), Phe290Asp, Phe290Asn and Phe290Ser (Alekseeva et al., Prot. Eng. Des. Select, 2012) in recombinant formate dehydrogenase from soya Glycine max (SoyFDH) lead to a significant (up to 30-100 times) increase in the thermal stability of the enzyme. The substitutions Phe290Asp, Phe290Asn and Phe290Ser were introduced into double mutant SoyFDH Ala267Met/Ile272Val by site-directed mutagenesis. Combinations of three substitutions did not lead to a noticeable change in the catalytic properties of the mutant enzymes. The stability of the resultant triple mutants was studied through thermal inactivation kinetics and differential scanning calorimetry. The thermal stability of the new mutant SoyFDHs was shown to be much higher than that of their precursors. The stability of the best mutant SoyFDH Ala267Met/Ile272Val/Phe290Asp turned out to be comparable to that of the most stable wild-type formate dehydrogenases from other sources. The results obtained with both methods indicate a great synergistic contribution of individual amino acid substitutions to the common stabilization effect.

scholarly journals Front Cover: Single Amino Acid Replacement in G‐7039 Leads to a 70‐fold Increase in Binding toward GHS‐R1a (ChemMedChem 20/2019)

ChemMedChem ◽  
2019 ◽  
Vol 14 (20) ◽  
pp. 1743-1743
Author(s):  
Tyler Lalonde ◽  
Milan M. Fowkes ◽  
Jinqiang Hou ◽  
Pierre E. Thibeault ◽  
Mark Milne ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fold Increase ◽  
Amino Acid Replacement ◽  
Single Amino Acid ◽  
Front Cover

Single Amino Acid Replacement in G‐7039 Leads to a 70‐fold Increase in Binding toward GHS‐R1a

ChemMedChem ◽  
2019 ◽  
Vol 14 (20) ◽  
pp. 1762-1766
Author(s):  
Tyler Lalonde ◽  
Milan M. Fowkes ◽  
Jinqiang Hou ◽  
Pierre E. Thibeault ◽  
Mark Milne ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fold Increase ◽  
Amino Acid Replacement ◽  
Single Amino Acid

scholarly journals Effects of thermal treatments on the stability of trans-resveratrol and yeast inactivation in trans-resveratrol-amplified grape juice

2014 ◽  
Vol 66 (1) ◽  
pp. 323-329 ◽  
Author(s):  
Dong-Un Lee ◽  
Hye Kim ◽  
Gu Lee ◽  
Seong-Ho Jeon ◽  
Jung-Jong Lee ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Grape Juice ◽  
Treatment Temperature ◽  
Inactivation Rate ◽  
Inactivation Kinetics ◽  
Decimal Reduction Time ◽  
First Order Kinetics ◽  
In Trans ◽  
The Stability ◽  
D Value

For the production of trans-resveratrol-amplified grape juice, both trans-resveratrol quantity and microbial quality are important for the functionality and shelf life of the juice. Therefore, the thermal stability of trans-resveratrol and thermal inactivation of Saccharomyces cerevisiae within the grape juice was investigated at 60?C, 80?C and 100?C. Inactivation of S. cerevisiae was fitted to first-order kinetics, and the inactivation rate (k1) and decimal reduction time (D-value) at each treatment temperature were estimated. The control grape juice had an inactivation rate of 0.0014 (s-1) and a D-value of 11.90 min at 60?C, whereas the trans-resveratrol-amplified grape juice had an inactivation rate of 0.0016 (s-1) and a Dvalue of 10.42 min at the same temperature. Similar inactivation kinetics of the control and trans-resveratrol-amplified grape juice were observed at 80?C and 100?C, which indicates that increased trans-resveratrol content does not affect the thermal inactivation of inoculated S. cerevisiae.

Improvement of the specificity of dipetarudin by site directed mutagenesis

2005 ◽  
Vol 93 (03) ◽  
pp. 430-436
Author(s):  
Katrin Mende ◽  
Goetz Nowak ◽  
Mercedes López
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
Amino Acid Replacement ◽  
Site Directed Mutagenesis ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Directed Mutagenesis ◽  
Chromogenic Assay ◽  
Wild Type Counterpart ◽  
Screening Assays

SummaryProtease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudin R10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudin R10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).

scholarly journals Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

2005 ◽  
Vol 71 (10) ◽  
pp. 5823-5827 ◽  
Author(s):  
Kazutoshi Fujii ◽  
Hirotaka Minagawa ◽  
Yoshinobu Terada ◽  
Takeshi Takaha ◽  
Takashi Kuriki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Random Mutagenesis ◽  
Hydrolytic Activity ◽  
Amino Acid Replacement ◽  
Site Directed Mutagenesis ◽  
Efficient Production ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Thermus Aquaticus ◽  
Hydrolytic Activities

ABSTRACT Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

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.

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