title: Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase.
abstract: The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP(+) analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP(+) in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.
authors.affiliation: Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Orru
authors.forename: Daniel E Torres
authors.initials: DE
authors.lastname: Pazmiño
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
mesh_terms: D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D009249:NADP; D006899:Mixed Function Oxygenases; D010100:Oxygen
references.citation: FEBS J. 2009 May;276(10):2833-40
references.pmid: 19459938
references.citation: J Pharmacol Exp Ther. 2007 Jan;320(1):266-73
references.pmid: 17050781
references.citation: Arch Biochem Biophys. 2007 Aug 15;464(2):251-9
references.pmid: 17531949
references.citation: Proc Natl Acad Sci U S A. 2008 May 6;105(18):6572-7
references.pmid: 18443301
references.citation: Biochem Biophys Res Commun. 2003 Jul 11;306(4):930-6
references.pmid: 12821131
references.citation: J Biol Chem. 1981 May 10;256(9):4611-8
references.pmid: 7217102
references.citation: J Biol Chem. 1983 Feb 10;258(3):1752-7
references.pmid: 6822532
references.citation: J Biol Chem. 1979 Jul 25;254(14):6449-55
references.pmid: 36396
references.citation: Trends Pharmacol Sci. 2008 Jun;29(6):294-301
references.pmid: 18423897
references.citation: Lancet. 1987 Mar 14;1(8533):634-5
references.pmid: 2881174
references.citation: Pharmacol Ther. 2005 Jun;106(3):357-87
references.pmid: 15922018
references.citation: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references.pmid: 15299374
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2184-95
references.pmid: 15572771
references.citation: Drug Metab Rev. 2002 Aug;34(3):503-11
references.pmid: 12214662
references.citation: Trends Plant Sci. 2007 Sep;12(9):412-8
references.pmid: 17765596
references.citation: Pharmacogenetics. 2000 Dec;10(9):799-807
references.pmid: 11191884
references.citation: Annu Rev Pharmacol Toxicol. 2006;46:65-100
references.pmid: 16402899
references.citation: Trends Biochem Sci. 2006 May;31(5):276-83
references.pmid: 16600599
references.citation: J Biol Chem. 1980 May 10;255(9):3817-9
references.pmid: 7372647
references.citation: J Biol Chem. 2005 Dec 16;280(50):41360-5
references.pmid: 16223729
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: J Biol Chem. 1981 May 10;256(9):4619-25
references.pmid: 7217103
references.citation: J Biol Chem. 1994 Sep 9;269(36):22459-62
references.pmid: 8077188
references.citation: BMC Biochem. 2005 Nov 25;6:26
references.pmid: 16309555
references.citation: J Am Chem Soc. 1977 Oct 26;99(22):7272-86
references.pmid: 915151
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):470-8
references.pmid: 20383000
references.citation: Nat Genet. 1997 Dec;17(4):491-4
references.pmid: 9398858
references.citation: Angew Chem Int Ed Engl. 2005 Mar 4;44(11):1704-6
references.pmid: 15693045