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

A A Pometun; P D Parshin; N P Galanicheva; L A Shaposhnikov; D L Atroshenko; E V Pometun; V V Burmakin; S Yu Kleymenov; S S Savin; V I Tishkov

doi:10.32607/actanaturae.11665

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

Acta Naturae. 2022 Jan-Mar;14(1):82-91. doi: 10.32607/actanaturae.11665.

Authors

A A Pometun^{1

2

3}, P D Parshin^{2

3}, N P Galanicheva², L A Shaposhnikov², D L Atroshenko^{1

2

3}, E V Pometun⁴, V V Burmakin², S Yu Kleymenov^{1

5}, S S Savin^{2

3}, V I Tishkov^{1

2

3}

Affiliations

¹ Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, 119071 Russia.
² Lomonosov Moscow State University, Department of Chemistry, Moscow, 119991 Russia.
³ Innovations and High Technologies MSU Ltd., Moscow, 109559 Russia.
⁴ Sechenov First Moscow State Medical University, Moscow, 119991 Russia.
⁵ Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, 119334 Russia.

Abstract

Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC 1.2.1.2) is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.

Keywords: Pseudomonas sp. 101; catalytic properties; formate dehydrogenase; site-directed mutagenesis; thermal stability.