title: Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.
abstract: l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.
authors: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA; Department of Chemistry, Faculty of Science, Damietta University, Damietta, 34517, Egypt.
authors: Heba
authors: H
authors: Abdelwahab
authors: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
authors: Reeder
authors: R
authors: Robinson
authors: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA.
authors: Pedro
authors: P
authors: Rodriguez
authors: Department of Chemistry, Faculty of Science, Damietta University, Damietta, 34517, Egypt.
authors: Camelia
authors: C
authors: Adly
authors: Department of Food Technology, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications, Alexandria, 21934, Egypt.
authors: Sohby
authors: S
authors: El-Sohaimy
authors: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA; Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 24061, USA; Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA. Electronic address: psobrado@vt.edu.
authors: Pablo
authors: P
authors: Sobrado
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D005415:Flavins / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006900:Hydroxylation; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008968:Molecular Conformation; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000737:chemistry*; D009615:Nocardia / Q000201:enzymology*; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D005415:Flavins; D009243:NAD; D009249:NADP; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C047196:lysine N-epsilon hydroxylase; D008239:Lysine; D010100:Oxygen
keywords: C4a-hydroperoxyflavin; Flavin-dependent monooxygenases; N-hydroxylating monooxygenases; Nocobactin; Siderophores; Virulence factor; l-lysine hydroxylase