The role of ala198 in the stability and coenzyme specificity of bacterial formate dehydrogenases

Abstract

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 ψ and φ. 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 KM (NAD+) value from 60 to 35 and from 80 to 45 μM for PseFDH and MorFDH, respectively, while the KM (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 μM 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(+).

Keywords: coenzyme specificity; kinetic parameters; site-directed mutagenesis; thermal stability.

Fig. 1

Alignment of amino acid sequences of formate dehydrogenases from different sources in the region of the coenzyme-binding domain. Bacterial FDHs are marked in blue: PseFDH –Pseudomonas sp.101 (UniProtKB/Swiss- Prot: P33160.3), MorFDH – Moraxella sp. C-1 (GenBank Accession Y13245), MycFDH –Mycobacterium vaccae N10 (GenBank BAB69476), HypFDH – Hyphomicrobium strain JC-17 [GenBank BAB55449], SmeFDH – Sinorhizobium meliloti 16262453 (GenBank NP_435497), BstFDH – Burkholderia stabilis (GenBank CP000378), SauFDH – Staphylococcus aureus (NCBI Reference Sequence: WP_031923037.1). FDHs from yeasts are marked in brown: CboFDH – Candida boidinii (GenBank Accession ABE69165), SceFDH – baker’s yeast S.cerevisiae (EMBL Z75296). FDHs from fungi are marked in magenta: MgrFDH –Mycosphaerella graminicola (Septoria tritici) (UniProt Q9Y790), MagFDH –Magnaporthe grisea (EMBL AA415108), NeuFDH –Neurospora crassa [GenBank Accession XP_961202.] AjcFDH –Ajellomyces capsulatus [GenBank Accession XP_001539240], GzeFDH –Gibberella zeae (GenBank Accession XM_386303) and FDHs from plants are marked in green: SoyFDH –soybean Glycine max (GenBank Accession GB BT094321), AthFDH –Arabidopsis thaliana (EMBL AF208029), QroFDH –oak Quercus robur (GenBank Accession GB AJ577266)

Fig. 2

Ramachandran plot for the structures of the apo-forms of formate dehydrogenases from bacteria Pseudomonas sp.101 (A) (PDB2NAC) and Moraxella sp. C-1 (B) (PDB3FN4). Only one pair of angles ψ and φ is shown for MorFDH because the crystallographic cell of the latter enzyme contains only one enzyme subunit

Fig. 3

Fragments of the structures of formate dehydrogenase from bacterium Pseudomonas sp.101. A – wild-type enzyme (complex with NAD⁺ and azide – 2NAD), B – model structure of the mutant PseFDH A198G (complex with NAD⁺); C and D – model structures of mutant PseFDH D221S and PseFDH A198G/D221S (both – complexes with NADP⁺), respectively

Fig. 4

Residual activity as a function of time for mutant PseFDHs and wild-type enzyme, 0.1 M phosphate buffer, pH 7.0, 63oC

Fig. 5

Temperature dependence of the inactivation rate constant in coordinates ln(k_in/T) ‑ 1/T for mutant PseFDHs and the wild-type enzyme