Effect of the C-terminal sequence on the catalytic properties and thermal stability of bacterial formate dehydrogenase

Abstract

Nicotinamide adenine dinucleotide (NAD⁺)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 (PseFDH) is the most stable enzyme among natural orthologs and is widely used in the synthesis of chiral compounds with NAD(P)⁺-dependent oxidoreductases. PseFDH is a homodimer and belongs to the subfamily of classic bacterial formate dehydrogenases with a polypeptide chain length of 400 amino acids. In the apo form of PseFDH and its complex with formate (PDB_2NAC and PDB_2GUG, respectively), only 374 residues are visible in each subunit, and in the structure of the holo form (PseFDH—NAD⁺—azide ternary complex, PDB_2NAD), 391 and 383 residues are visible in two subunits. The amino acid sequencing of PseFDH isolated from the original strain Pseudomonas sp. 101, showed that the subunit in this enzyme consists of 393 residues. We prepared the full-length recombinant PseFDH consisting of 400 residues and truncated mutants consisting of 393, 383, and 374 residues (PseFDH_400, PseFDH_393, PseFDH_383, and PseFDH_374, respectively) and studied their properties. Our results and analysis of the structure of the holo form of FDH from Moraxella sp.C1 (PDB_2GSD) indicate that shortening of the C-terminus of PseFDH during isolation from the natural strain and the presence of 391, 383, and 374 residue subunits in the crystals are caused by proteolysis.