title: Mutagenesis of the "leucine gate" to explore the basis of catalytic versatility in soluble methane monooxygenase.
abstract: Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent nonheme oxygenase that naturally oxidizes methane to methanol and can also cooxidize a wide range of adventitious substrates, including mono- and diaromatic hydrocarbons. Leucine 110, at the mouth of the active site in the alpha subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control the access of substrates to the active site. Previous crystallography of the wild-type sMMO has indicated at least two conformations of the enzyme that have the "leucine gate" open to different extents, and mutagenesis of homologous enzymes has indicated a role for this residue in the control of substrate range and regioselectivity with aromatic substrates. By further refinement of the system for homologous expression of sMMO that we developed previously, we have been able to prepare a range of site-directed mutations at position 110 in the alpha subunit of sMMO. All the mutants (with Gly, Cys, Arg, and Tyr, respectively, at this position) showed relaxations of regioselectivity compared to the wild type with monoaromatic substrates and biphenyl, including the appearance of new products arising from hydroxylation at the 2- and 3- positions on the benzene ring. Mutants with the larger Arg and Trp residues at position 110 also showed shifts in regioselectivity during naphthalene hydroxylation from the 2- to the 1- position. No evidence that mutagenesis of Leu 110 could allow very large substrates to enter the active site was found, however, since the mutants (like the wild type) were inactive toward the triaromatic hydrocarbons anthracene and phenanthrene. Thus, our results indicate that the "leucine gate" in sMMO is more important in controlling the precision of regioselectivity than the sizes of substrates that can enter the active site.
authors: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom.
authors: Elena
authors: E
authors: Borodina
authors: Tim
authors: T
authors: Nichol
authors: Marc G
authors: MG
authors: Dumont
authors: Thomas J
authors: TJ
authors: Smith
authors: J Colin
authors: JC
authors: Murrell
mesh_terms: D002384:Catalysis; D007930:Leucine / Q000737:chemistry / Q000235:genetics*; D020602:Methylosinus trichosporium / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D009281:Naphthalenes; D011994:Recombinant Proteins; C031721:naphthalene; D010105:Oxygenases; C021555:methane monooxygenase; D007930:Leucine
references: Gene. 1994 Jul 22;145(1):69-73
references: 8045426
references: Proteins. 1997 Oct;29(2):141-52
references: 9329079
references: Appl Environ Microbiol. 1995 Oct;61(10):3549-55
references: 7486989
references: J Bacteriol. 2004 Jul;186(14):4705-13
references: 15231803
references: Microbiology. 1999 Feb;145 ( Pt 2):461-70
references: 10075428
references: Nature. 1980 Aug 7;286(5773):561-4
references: 6772967
references: Nature. 1993 Dec 9;366(6455):537-43
references: 8255292
references: Structure. 1998 May 15;6(5):571-86
references: 9634695
references: FEMS Microbiol Rev. 2003 Oct;27(4):449-79
references: 14550940
references: Anal Biochem. 1984 Feb;137(1):266-7
references: 6329026
references: Arch Microbiol. 2000 May-Jun;173(5-6):325-32
references: 10896210
references: Arch Microbiol. 1999 May-Jun;171(6):364-70
references: 10369892
references: J Biol Chem. 1993 Oct 15;268(29):21569-77
references: 8408008
references: Anal Biochem. 1983 Jul 1;132(1):6-13
references: 6312838
references: J Bacteriol. 2000 Mar;182(6):1641-9
references: 10692370
references: Biotechnol Bioeng. 1996 Aug 5;51(3):349-59
references: 18624367
references: Gene. 1982 Oct;19(3):259-68
references: 6295879
references: J Bacteriol. 2006 Jul;188(13):4962-9
references: 16788204
references: Protein Sci. 1997 Mar;6(3):556-68
references: 9070438
references: Appl Environ Microbiol. 1994 Jul;60(7):2473-82
references: 8074526
references: J Gen Microbiol. 1992 Jul;138(7):1301-7
references: 1512560
references: Appl Environ Microbiol. 1998 Jul;64(7):2710-5
references: 9647854
references: Microbiology. 2003 Jul;149(Pt 7):1771-84
references: 12855729
references: Arch Microbiol. 2000 Jul-Aug;174(1-2):35-41
references: 10985740
references: J Biol Chem. 2002 Feb 22;277(8):5858-65
references: 11709550
references: Appl Environ Microbiol. 2004 Jun;70(6):3253-62
references: 15184119
references: Appl Environ Microbiol. 2004 Jun;70(6):3222-31
references: 15184115
references: Appl Environ Microbiol. 2002 Nov;68(11):5265-73
references: 12406713
references: J Bacteriol. 2002 Jan;184(2):344-9
references: 11751810
references: Gene. 1995 Dec 1;166(1):175-6
references: 8529885
references: Gene. 1989 Apr 15;77(1):51-9
references: 2744487
references: Science. 1997 Jan 24;275(5299):515-8
references: 8999792
references: J Biol Chem. 2004 Jul 16;279(29):30600-10
references: 15096510
references: Mol Microbiol. 1991 Feb;5(2):335-42
references: 1904125
references: Biodegradation. 1990;1(1):19-29
references: 1368139
references: J Mol Biol. 2000 Feb 18;296(2):701-12
references: 10669618