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

Abstract

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.

Authors:
 [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:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1239772
Report Number(s):
BNL-108294-2015-JA
Journal ID: ISSN 0021-9258; R&D Project: LS001
Grant/Contract Number:  
SC00112704
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 20; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; lysine monooxygenase; flavin-dependent monooxygenases; N-hydroxylating monooxygenases; siderophore; virulence factor; C4a-hydroperoxyflavin

Citation Formats

Binda, Claudia, Robinson, Reeder M., Martin del Campo, Julia S., Keul, Nicholas D., Rodriguez, Pedro J., Robinson, Howard H., Mattevi, Andrea, and Sobrado, Pablo. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation. United States: N. p., 2015. Web. doi:10.1074/jbc.M114.629485.
Binda, Claudia, Robinson, Reeder M., Martin del Campo, Julia S., Keul, Nicholas D., Rodriguez, Pedro J., Robinson, Howard H., Mattevi, Andrea, & Sobrado, Pablo. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation. United States. https://doi.org/10.1074/jbc.M114.629485
Binda, Claudia, Robinson, Reeder M., Martin del Campo, Julia S., Keul, Nicholas D., Rodriguez, Pedro J., Robinson, Howard H., Mattevi, Andrea, and Sobrado, Pablo. 2015. "An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation". United States. https://doi.org/10.1074/jbc.M114.629485. https://www.osti.gov/servlets/purl/1239772.
@article{osti_1239772,
title = {An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation},
author = {Binda, Claudia and Robinson, Reeder M. and Martin del Campo, Julia S. and Keul, Nicholas D. and Rodriguez, Pedro J. and Robinson, Howard H. and Mattevi, Andrea and Sobrado, Pablo},
abstractNote = {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.},
doi = {10.1074/jbc.M114.629485},
url = {https://www.osti.gov/biblio/1239772}, journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 20,
volume = 290,
place = {United States},
year = {Mon Mar 23 00:00:00 EDT 2015},
month = {Mon Mar 23 00:00:00 EDT 2015}
}

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Cited by: 34 works
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Works referenced in this record:

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Works referencing / citing this record:

Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis
journal, October 2017


Crystal structure of pyrrolizidine alkaloid N -oxygenase from the grasshopper Zonocerus variegatus
journal, April 2018


Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C -Nucleoside Scaffolds
journal, November 2019


l -lysine metabolism to N -hydroxypipecolic acid: an integral immune-activating pathway in plants
journal, September 2018


Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus
text, January 2018