Structure of the monooxygenase component of a two-component flavoprotein monooxygenase
- Andrea Alfieri*,
- Francesco Fersini*,
- Nantidaporn Ruangchan†,
- Methinee Prongjit†,
- Pimchai Chaiyen†,‡, and
- Andrea Mattevi*,‡
- *Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy; and
- †Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
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Edited by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved November 29, 2006 (received for review September 23, 2006)
Abstract
p-Hydroxyphenylacetate hydroxylase from Acinetobacter baumannii is a two-component system consisting of a NADH-dependent FMN reductase and a monooxygenase (C2) that uses reduced FMN as substrate. The crystal structures of C2 in the ligand-free and substrate-bound forms reveal a preorganized pocket that binds reduced FMN without large conformational changes. The Phe-266 side chain swings out to provide the space for binding p-hydroxyphenylacetate that is oriented orthogonal to the flavin ring. The geometry of the substrate-binding site of C2 is significantly different from that of p-hydroxybenzoate hydroxylase, a single-component flavoenzyme that catalyzes a similar reaction. The C2 overall structure resembles the folding of medium-chain acyl-CoA dehydrogenase. An outstanding feature in the C2 structure is a cavity located in front of reduced FMN; it has a spherical shape with a 1.9-Å radius and a 29-Å3 volume and is interposed between the flavin C4a atom and the substrate atom to be hydroxylated. The shape and position of this cavity are perfectly fit for housing the oxygen atoms of the flavin C4a-hydroperoxide intermediate that is formed upon reaction of the C2-bound reduced flavin with molecular oxygen. The side chain of His-396 is predicted to act as a hydrogen-bond donor to the oxygen atoms of the intermediate. This architecture promotes the nucleophilic attack of the substrate onto the terminal oxygen of the hydroperoxyflavin. Comparative analysis with the structures of other flavoenzymes indicates that a distinctive feature of monooxygenases is the presence of specific cavities that encapsulate and stabilize the crucial hydroperoxyflavin intermediate.
Footnotes
- ‡To whom correspondence may be addressed. E-mail: mattevi{at}ipvgen.unipv.it or scpcy{at}mucc.mahidol.ac.th
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Author contributions: P.C. and A.M. designed research; A.A., F.F., N.R., and M.P. performed research; A.A., P.C., and A.M. analyzed data; and A.A., P.C., and A.M. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS direct submission.
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Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2JBR, 2JBT, and 2JBS).
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This article contains supporting information online at www.pnas.org/cgi/content/full/0608381104/DC1.
- Abbreviations:
- C1,
- reductase component of HPA hydroxylase;
- C2,
- monooxygenase component of HPA hydroxylase;
- FMNH−,
- two-electron reduced FMN;
- HPA,
- p-hydroxyphenylacetate.
- © 2007 by The National Academy of Sciences of the USA
