Engineering the Active Site of Formate Dehydrogenase from Staphylococcus aureus: Introduction of the Additional Loop and Histigine Residues to the Structure

Abstract

NAD⁺-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5–3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in $K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$; the value of k_cat has not changed. When T250H is replaced, an increase in $K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$ is observed, k_cat decreases from 20 to 13 s^–1. The replacement of K368H led to a slight increase in $K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$, k_cat decreased from 20 to 6 s^–1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in $K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$ and $K_{{\text{M}}}^{{{\text{HCO}}{{{\text{O}}}^{ - }}}}$. A bigger effect was observed for $K_{{\text{M}}}^{{{\text{NA}}{{{\text{D}}}^{{\text{ + }}}}}}$—the difference was more than 10 times. For mutant SauFDH with insertions k_cat significantly reduced to 4 s^–1. Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH.