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Title: An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.
 [1] ;  [2] ;  [2] ;  [2] ;  [2] ;  [3] ;  [1] ;  [2]
  1. Univ. of Pavia, Pavia (Italy)
  2. Virginia Tech, Blacksburg, VA (United States)
  3. Brookhaven National Lab. (BNL), Upton, NY (United States)
Publication Date:
Report Number(s):
Journal ID: ISSN 0021-9258; R&D Project: LS001
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 20; Journal ID: ISSN 0021-9258
American Society for Biochemistry and Molecular Biology
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
36 MATERIALS SCIENCE; lysine monooxygenase; flavin-dependent monooxygenases; N-hydroxylating monooxygenases; siderophore; virulence factor; C4a-hydroperoxyflavin
OSTI Identifier: