Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

• Published in: Applied microbiology and biotechnology 2014-08, Vol.98 (16), p.7081-7088
• Main Authors: Gao, Hui, Tiwari, Manish Kumar, Singh, Raushan Kumar, Sung, Bong Hyun, Kim, Sun Chang, Lee, Jung-Kul
• Format: Article
• Language: English
• Subjects: adenine; Amino Acid Substitution; Analysis; arginine; Biocatalysts; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; catalytic activity; E coli; Enzymatic activity; enzyme activity; Enzyme kinetics; Enzyme Stability; Enzymes; Flavoproteins - chemistry; Flavoproteins - genetics; Flavoproteins - metabolism; Genes; half life; Life Sciences; lysine; Lysine - genetics; Microbial Genetics and Genomics; Microbiology; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; mutants; Mutation; Mutation, Missense; NAD (coenzyme); NAD - metabolism; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; Oxidases; Oxidation; Oxidation-Reduction; oxygen; Oxygen - metabolism; Physiological aspects; Plasmids; Point Mutation; Protein expression; Proteins; Residues; Streptococcus pyogenes; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics; Studies; sugars; Temperature; thermal stability; Water - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-014-5666-y

Nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes (SpNox) is a flavoprotein harboring one molecule of noncovalently bound flavin adenine dinucleotide. It catalyzes the oxidation of NADH by reducing molecular O₂ to H₂O directly through a four-electron reduction. In this study, we selected the lysine residues on the surface of SpNox and mutated them into arginine residues to study the effect on the enzyme activity. A single-point mutation (K184R) at the surface of SpNox enhanced NADH oxidase activity by approximately 50 % and improved thermostability with 46.6 % longer half life at 30 °C. Further insights into the function of residue K184 were obtained by substituting it with other nonpolar, polar, positively charged, and negatively charged residues. To elucidate the role of this residue, computer-assisted molecular modeling and substrate docking were performed. The results demonstrate that even a single mutation at the surface of the enzyme induces changes in the interaction at the active site and affects the activity and stability. Additionally, the data also suggest that the K184R mutant can be used as an effective biocatalyst for NAD⁺ regeneration in L-rare sugar production.