ENGINEERING THE ACTIVE SITE OF FORMATE DEHYDROGE-NASE FROM STAPHYLOCOCCUS AUREUS: INTRODUCTION TO THE STRUCTURE OF THE ADDITIONAL LOOP AND HISTIDINE RESIDUES

Abstract

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs signi cantly 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) speci c activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of threedimensional structures and the alignment of amino acid sequences, substitutions promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in KMNAD+; the value of kcat has not changed. When Т250Н is replaced, an increase in KMNAD+ is observed, kcat decreases from 20 to 13 s-1. The replacement of K368H led to a slight increase in KMNAD+, kcat decreased from 20 s-1 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 KMNAD+ and KMHCOO-. A bigger effect was observed for KMNAD+ - the difference was more than 10 times. For mutant SauFDH with insertions kcat signi cantly reduced to 4 s-1. Similar results were observed for mutants with multipoint substitutions. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH.