TY - JOUR

T1 - Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

AU - Gao, Hui

AU - Tiwari, Manish Kumar

AU - Singh, Raushan Kumar

AU - Sung, Bong Hyun

AU - Kim, Sun Chang

AU - Lee, Jung-Kul

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Amino Acid Substitution

KW - Enzyme Stability

KW - Flavoproteins

KW - Lysine

KW - Multienzyme Complexes

KW - Mutagenesis, Site-Directed

KW - Mutant Proteins

KW - Mutation, Missense

KW - NAD

KW - NADH, NADPH Oxidoreductases

KW - Oxidation-Reduction

KW - Oxygen

KW - Point Mutation

KW - Streptococcus pyogenes

KW - Temperature

KW - Water

U2 - 10.1007/s00253-014-5666-y

DO - 10.1007/s00253-014-5666-y

M3 - Journal article

C2 - 24687749

VL - 98

SP - 7081

EP - 7088

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 16

ER -