title: Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida.
abstract: To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.
authors.affiliation: Departamento de Protección Ambiental, Estación del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
authors.forename: Pieter
authors.initials: P
authors.lastname: van Dillewijn
authors.forename: Rolf-Michael
authors.initials: RM
authors.lastname: Wittich
authors.forename: Antonio
authors.initials: A
authors.lastname: Caballero
authors.forename: Juan-Luis
authors.initials: JL
authors.lastname: Ramos
mesh_terms: D003001:Cloning, Molecular; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D053858:Metabolic Networks and Pathways; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010802:Phylogeny; D016958:Pseudomonas putida / Q000201:enzymology*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014303:Trinitrotoluene / Q000378:metabolism
chemical_list: D005420:Flavoproteins; D009573:Nitrites; D000644:Quaternary Ammonium Compounds; D014303:Trinitrotoluene; D009252:NADPH Dehydrogenase; D014166:Transferases
references.citation: J Bacteriol. 2005 Sep;187(17):5937-45
references.pmid: 16109935
references.citation: J Biol Chem. 2003 May 30;278(22):19891-7
references.pmid: 12660247
references.citation: J Bacteriol. 1999 Oct;181(20):6254-63
references.pmid: 10515912
references.citation: Environ Microbiol. 2007 Jun;9(6):1535-40
references.pmid: 17504490
references.citation: Biochem J. 2006 Feb 15;394(Pt 1):335-44
references.pmid: 16293111
references.citation: Environ Microbiol. 2005 Aug;7(8):1211-9
references.pmid: 16011758
references.citation: Appl Environ Microbiol. 1998 Jan;64(1):246-52
references.pmid: 16349484
references.citation: Annu Rev Genet. 2004;38:615-43
references.pmid: 15568988
references.citation: J Mol Biol. 2007 Jun 8;369(3):829-40
references.pmid: 17466326
references.citation: J Biol Chem. 2005 May 27;280(21):20887-93
references.pmid: 15767250
references.citation: J Biol Chem. 1999 Apr 2;274(14):9357-62
references.pmid: 10092614
references.citation: J Biol Chem. 1998 Dec 4;273(49):32753-62
references.pmid: 9830019
references.citation: Nature. 2006 Mar 16;440(7082):341-5
references.pmid: 16541074
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: J Bacteriol. 1989 Dec;171(12):6782-90
references.pmid: 2687253
references.citation: Environ Microbiol. 2002 Dec;4(12):799-808
references.pmid: 12534463
references.citation: Nature. 2007 Oct 11;449(7163):677-81
references.pmid: 17928853
references.citation: Curr Opin Biotechnol. 2005 Jun;16(3):275-81
references.pmid: 15961028
references.citation: Science. 2001 Dec 7;294(5549):2158-63
references.pmid: 11739955
references.citation: Appl Environ Microbiol. 2004 Jun;70(6):3566-74
references.pmid: 15184158
references.citation: Environ Microbiol. 2006 Jul;8(7):1306-10
references.pmid: 16817939
references.citation: Environ Sci Technol. 2008 Feb 1;42(3):734-9
references.pmid: 18323095
references.citation: Appl Microbiol Biotechnol. 2007 Aug;76(2):267-77
references.pmid: 17534614
references.citation: Appl Environ Microbiol. 2003 Mar;69(3):1542-7
references.pmid: 12620841
references.citation: J Mol Biol. 2006 Aug 4;361(1):140-52
references.pmid: 16822524
references.citation: Protein Expr Purif. 2004 Aug;36(2):280-91
references.pmid: 15249051
references.citation: Appl Environ Microbiol. 1998 Aug;64(8):2864-8
references.pmid: 9687442
references.citation: J Bacteriol. 2005 Jan;187(1):396-9
references.pmid: 15601726
references.citation: Microbiology (Reading). 2002 Jun;148(Pt 6):1607-1614
references.pmid: 12055282
references.citation: Appl Environ Microbiol. 2000 Nov;66(11):4742-50
references.pmid: 11055918
references.citation: J Biol Chem. 2005 Jul 29;280(30):27904-13
references.pmid: 15890652
references.citation: Appl Environ Microbiol. 2008 Nov;74(21):6820-3
references.pmid: 18791007