Assessment of Formate Dehydrogenase Stress Stability in vivo using Inactivation by Hydrogen Peroxide

Abstract

Kinetic studies on hydrogen peroxide–induced inactivation of mutant formate dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of hydrogen peroxide, the decrease in enzyme activity follows first–order kinetics. Therefore, the first–order effective inactivation kinetic constants determined for various FDH forms at a constant H2O2 concentration can be used as a quantitative measure of the enzyme stability. It was shown that two cysteine residues located in the active site formate– and coenzyme–binding domains (Cys145 and Cys255, respectively) make similar contributions to the enzyme stability, while the contribution of Cys354 is insignificant. The inactivation kinetics of wild–type PseFDH, mutant PseFDH Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium Staphylococcus aureus, higher plants Arabidopsis thaliana, and soya Glycine max, was studied. It was found that the stress–induced FDHs are at least 20 times more stable than the nonstress–induced PseFDH from Pseudomonas sp. 101 grown on methanol.

Co-immobilized Multienzyme System for the Cofactor-Driven Cascade Synthesis of (R)-2-Amino-3-(2-bromophenyl)propanoic Acid: A Model Reaction

“…The poor stability of native cb FDH was well documented in the literature, which was prone to thermal deactivation at >40 °C and chemical instability against chemicals, which can influence the stability of tertiary and quaternary structures. Cysteine residues are the most critical residues responsible for stability, and hence, oxidation of the cysteine thiol group possibly leads to enzyme deactivation . The use of PEI has been demonstrated to have a positive effect on enzyme activity and stability on enzymes like glucose dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, and β-galactosidase. − Lin et al have reported PEI-grafted graphene oxide as a novel scaffold for immobilization for cb FDH, which showed 47.4% activity after eight times repeated use, in addition to having 2.4-fold activity as compared to that of an enzyme immobilized on plain graphene oxide …”

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S

“…The C255A replacement results in preservation of 60% of the enzyme activity after 90-day storage at 25°C, whereas the wild-type PseFDH becomes completely inactive at this point [ 14 ]. The double replacement C145S/C255A decreases the enzyme inactivation rate constant in the presence of 100 mM hydrogen peroxide by almost 100 times [ 15 ]. The A198G replacement provides a decrease in structural tension in the polypeptide chain turn connecting βA beta-sheet and αB helix in the coenzyme binding domain of PseFDH active site.…”

“…The effect of N-terminal His-tag on the properties of the wild-type enzyme and its NADP + -specific mutants has been studied by site-directed mutagenesis [ 13 ]. Chemical stability of PseFDH has been improved as well, and the mutants with an increased stability in the presence of hydrogen peroxide have been produced [ 14 , 15 ].…”

In Vivo Selection for Formate Dehydrogenases with High Efficiency and Specificity toward NADP+

“…Moreover, mutation A198G improves the binding of NADP + when cofactor specificity has already changed: e.g., by the D221Q mutation . The third residue is C255, − which interacts with the adenine moiety of NAD + , and its mutation to valine was shown to improve the binding affinity toward NADP + . Similarly, the NADP + -dependent FDH from Burkholderia spp.…”