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

Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

doi:10.1074/jbc.M114.629485

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

Journal Article · Mon Mar 23 00:00:00 EDT 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M114.629485· OSTI ID:1239772

Binda, Claudia ^[1]; Robinson, Reeder M. ^[2]; Martin del Campo, Julia S. ^[2]; Keul, Nicholas D. ^[2]; Rodriguez, Pedro J. ^[2]; Robinson, Howard H. ^[3]; Mattevi, Andrea ^[1]; Sobrado, Pablo ^[2]

Univ. of Pavia, Pavia (Italy)
Virginia Tech, Blacksburg, VA (United States)
Brookhaven National Lab. (BNL), Upton, NY (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

OSTI ID:: 1239772

Alternate ID(s):: OSTI ID: 1228916
OSTI ID: 1228982

Report Number(s):: BNL--108294-2015-JA

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 20 Vol. 290; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

36 MATERIALS SCIENCE
C4a-hydroperoxyflavin
N-hydroxylating monooxygenases
flavin-dependent monooxygenases
lysine monooxygenase
siderophore
virulence factor

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

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Cited By (6)

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