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:
-
- Univ. of Pavia, Pavia (Italy)
- Virginia Tech, Blacksburg, VA (United States)
- 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}
}
Web of Science
Works referenced in this record:
Enzymatic Tailoring of Ornithine in the Biosynthesis of the Rhizobium Cyclic Trihydroxamate Siderophore Vicibactin
journal, October 2009
- Heemstra, John R.; Walsh, Christopher T.; Sattely, Elizabeth S.
- Journal of the American Chemical Society, Vol. 131, Issue 42
HKL2MAP : a graphical user interface for macromolecular phasing with SHELX programs
journal, September 2004
- Pape, Thomas; Schneider, Thomas R.
- Journal of Applied Crystallography, Vol. 37, Issue 5
Linking Crystallographic Model and Data Quality
journal, May 2012
- Karplus, P. A.; Diederichs, K.
- Science, Vol. 336, Issue 6084
Presenting your structures: the CCP 4 mg molecular-graphics software
journal, March 2011
- McNicholas, S.; Potterton, E.; Wilson, K. S.
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent N -Hydroxylase
journal, September 2014
- Setser, Jeremy W.; Heemstra, John R.; Walsh, Christopher T.
- Biochemistry, Vol. 53, Issue 38
Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997
- Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
- Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
Overview of the CCP 4 suite and current developments
journal, March 2011
- Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
On the Estimation of Bound Hydroxylamine in Biological Materials.
journal, January 1948
- Csáky, Tihamér Z.; Hassel, O.; Rosenberg, Thomas
- Acta Chemica Scandinavica, Vol. 2
Autoinduction of Protein Expression
journal, April 2009
- Fox, Brian G.; Blommel, Paul G.
- Current Protocols in Protein Science, Vol. 56, Issue 1
Genetic Organization of the Region Encoding Regulation, Biosynthesis, and Transport of Rhizobactin 1021, a Siderophore Produced by Sinorhizobium meliloti
journal, April 2001
- Lynch, D.; O'Brien, J.; Welch, T.
- Journal of Bacteriology, Vol. 183, Issue 8
Aspergillus fumigatus SidA Is a Highly Specific Ornithine Hydroxylase with Bound Flavin Cofactor
journal, August 2010
- Chocklett, Samuel W.; Sobrado, Pablo
- Biochemistry, Vol. 49, Issue 31
Two Structures of an N -Hydroxylating Flavoprotein Monooxygenase : ORNITHINE HYDROXYLASE FROM PSEUDOMONAS AERUGINOSA
journal, July 2011
- Olucha, Jose; Meneely, Kathleen M.; Chilton, Annemarie S.
- Journal of Biological Chemistry, Vol. 286, Issue 36
Snapshots of Enzymatic Baeyer-Villiger Catalysis: OXYGEN ACTIVATION AND INTERMEDIATE STABILIZATION
journal, June 2011
- Orru, Roberto; Dudek, Hanna M.; Martinoli, Christian
- Journal of Biological Chemistry, Vol. 286, Issue 33
Mechanistic and structural studies of the N-hydroxylating flavoprotein monooxygenases
journal, December 2011
- Olucha, Jose; Lamb, Audrey L.
- Bioorganic Chemistry, Vol. 39, Issue 5-6
Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes.
journal, August 1996
- Kang, H. Y.; Brickman, T. J.; Beaumont, F. C.
- Journal of Bacteriology, Vol. 178, Issue 16
Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca
journal, August 2010
- Dudek, Hanna M.; Torres Pazmiño, Daniel E.; Rodríguez, Cristina
- Applied Microbiology and Biotechnology, Vol. 88, Issue 5
Identification of a Cluster of Genes that Directs Desferrioxamine Biosynthesis in Streptomyces c oelicolor M145
journal, December 2004
- Barona-Gómez, Francisco; Wong, Ursula; Giannakopulos, Anastassios E.
- Journal of the American Chemical Society, Vol. 126, Issue 50
Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase
journal, June 2012
- Romero, Elvira; Fedkenheuer, Michael; Chocklett, Samuel W.
- Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1824, Issue 6
Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA
journal, April 2014
- Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael
- Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1844, Issue 4
Toward rational protein crystallization: A Web server for the design of crystallizable protein variants
journal, August 2007
- Goldschmidt, Lukasz; Cooper, David R.; Derewenda, Zygmunt S.
- Protein Science, Vol. 16, Issue 8
Kinetic Mechanism of Ornithine Hydroxylase (PvdA) from Pseudomonas aeruginosa : Substrate Triggering of O 2 Addition but Not Flavin Reduction
journal, May 2009
- Meneely, Kathleen M.; Barr, Eric W.; Bollinger, J. Martin
- Biochemistry, Vol. 48, Issue 20
Comprehensive Spectroscopic, Steady State, and Transient Kinetic Studies of a Representative Siderophore-associated Flavin Monooxygenase
journal, July 2010
- Mayfield, Jeffery A.; Frederick, Rosanne E.; Streit, Bennett R.
- Journal of Biological Chemistry, Vol. 285, Issue 40
iMOSFLM : a new graphical interface for diffraction-image processing with MOSFLM
journal, March 2011
- Battye, T. Geoff G.; Kontogiannis, Luke; Johnson, Owen
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
Substrate Binding Modulates the Activity of Mycobacterium smegmatis G, a Flavin-Dependent Monooxygenase Involved in the Biosynthesis of Hydroxamate-Containing Siderophores
journal, October 2011
- Robinson, Reeder; Sobrado, Pablo
- Biochemistry, Vol. 50, Issue 39
A Method of Preparation and Purification of (4R)-Deuterated-Reduced Nicotinamide Adenine Dinucleotide Phosphate
journal, September 1994
- Jeong, S. S.; Gready, J. E.
- Analytical Biochemistry, Vol. 221, Issue 2
Coot model-building tools for molecular graphics
journal, November 2004
- Emsley, Paul; Cowtan, Kevin
- Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
Structural Insight into the Mechanism of Oxygen Activation and Substrate Selectivity of Flavin-Dependent N-Hydroxylating Monooxygenases
journal, August 2012
- Franceschini, Stefano; Fedkenheuer, Michael; Vogelaar, Nancy J.
- Biochemistry, Vol. 51, Issue 36
Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase
journal, April 2008
- Alfieri, A.; Malito, E.; Orru, R.
- Proceedings of the National Academy of Sciences, Vol. 105, Issue 18
FAD and substrate analogs as probes for lysine N6-hydroxylase from Escherichia coli EN 222
journal, May 1993
- Macheroux, Peter; Plattner, Hans Jurgen; Romaguera, Antonio
- European Journal of Biochemistry, Vol. 213, Issue 3
The Aspergillus fumigatus Siderophore Biosynthetic Gene sidA, Encoding L-Ornithine N5-Oxygenase, Is Required for Virulence
journal, August 2005
- Hissen, A. H. T.; Wan, A. N. C.; Warwas, M. L.
- Infection and Immunity, Vol. 73, Issue 9
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
Identification of Nocobactin NA Biosynthetic Gene Clusters in Nocardia farcinica
journal, November 2010
- Hoshino, Y.; Chiba, K.; Ishino, K.
- Journal of Bacteriology, Vol. 193, Issue 2
Identification of the NAD(P)H Binding Site of Eukaryotic UDP-Galactopyranose Mutase
journal, October 2012
- Dhatwalia, Richa; Singh, Harkewal; Solano, Luis M.
- Journal of the American Chemical Society, Vol. 134, Issue 43
Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor
journal, July 2009
- Mirza, I. Ahmad; Yachnin, Brahm J.; Wang, Shaozhao
- Journal of the American Chemical Society, Vol. 131, Issue 25
Crystal structure of a Baeyer-Villiger monooxygenase
journal, August 2004
- Malito, E.; Alfieri, A.; Fraaije, M. W.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 36
Mechanistic studies on the flavin-dependent N6-lysine monooxygenase MbsG reveal an unusual control for catalysis
journal, May 2014
- Robinson, Reeder M.; Rodriguez, Pedro J.; Sobrado, Pablo
- Archives of Biochemistry and Biophysics, Vol. 550-551
Preparation of selenomethionyl proteins for phase determination
book, January 1997
- Doublié, Sylvie
- Methods in Enzymology
- p. 523-530
Systematic analysis of domain motions in proteins from conformational change: New results on citrate synthase and T4 lysozyme
journal, February 1998
- Hayward, Steven; Berendsen, Herman J. C.
- Proteins: Structure, Function, and Genetics, Vol. 30, Issue 2
Mechanistic Studies of Cyclohexanone Monooxygenase: Chemical Properties of Intermediates Involved in Catalysis †
journal, September 2001
- Sheng, Dawei; Ballou, David P.; Massey, Vincent
- Biochemistry, Vol. 40, Issue 37
Biochemical Characterization of a Flavin Adenine Dinculeotide-Dependent Monooxygenase, Ornithine Hydroxylase from Pseudomonas aeruginosa , Suggests a Novel Reaction Mechanism †
journal, October 2007
- Meneely, Kathleen M.; Lamb, Audrey L.
- Biochemistry, Vol. 46, Issue 42
Isolation and some properties of lysineN 6-hydroxylase fromEscherichia coli strain EN222
journal, January 1989
- Plattner, Hans-J�rgen; Pfefferle, Petra; Romaguera, Antonio
- Biology of Metals, Vol. 2, Issue 1
Kinetic Mechanism of Phenylacetone Monooxygenase from Thermobifida fusca †
journal, April 2008
- Torres Pazmiño, Daniel E.; Baas, Bert-Jan; Janssen, Dick B.
- Biochemistry, Vol. 47, Issue 13
The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages
journal, February 2000
- De Voss, J. J.; Rutter, K.; Schroeder, B. G.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 3
The oxidative half-reaction of liver microsomal FAD-containing monooxygenase.
journal, May 1981
- Beaty, N. B.; Ballou, D. P.
- Journal of Biological Chemistry, Vol. 256, Issue 9
Works referencing / citing this record:
Characterization of the flavoenzyme XiaK as an N-hydroxylase and implications in indolosesquiterpene diversification
journal, January 2017
- Zhang, Qingbo; Li, Huixian; Yu, Lu
- Chemical Science, Vol. 8, Issue 7
Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis
journal, October 2017
- Bufkin, Kendra; Sobrado, Pablo
- Molecules, Vol. 22, Issue 10
Crystal structure of pyrrolizidine alkaloid N -oxygenase from the grasshopper Zonocerus variegatus
journal, April 2018
- Kubitza, Christian; Faust, Annette; Gutt, Miriam
- Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 5
Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C -Nucleoside Scaffolds
journal, November 2019
- Zhang, Meng; Zhang, Peichao; Xu, Gudan
- Applied and Environmental Microbiology, Vol. 86, Issue 2
l -lysine metabolism to N -hydroxypipecolic acid: an integral immune-activating pathway in plants
journal, September 2018
- Hartmann, Michael; Zeier, Jürgen
- The Plant Journal, Vol. 96, Issue 1
Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus
text, January 2018
- Kubitza, Christian; Faust, Annette; Gutt, Miriam
- Deutsches Elektronen-Synchrotron, DESY, Hamburg