Mutagenesis of the "leucine gate" to explore the basis of catalytic versatility in soluble methane monooxygenase.
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.
Borodina
Elena
E
Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom.
Nichol
Tim
T
Dumont
Marc G
MG
Smith
Thomas J
TJ
Murrell
J Colin
JC
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
D009281:Naphthalenes; D011994:Recombinant Proteins; C031721:naphthalene; D010105:Oxygenases; C021555:methane monooxygenase; D007930:Leucine
Gene. 1994 Jul 22;145(1):69-73
8045426
Proteins. 1997 Oct;29(2):141-52
9329079
Appl Environ Microbiol. 1995 Oct;61(10):3549-55
7486989
J Bacteriol. 2004 Jul;186(14):4705-13
15231803
Microbiology. 1999 Feb;145 ( Pt 2):461-70
10075428
Nature. 1980 Aug 7;286(5773):561-4
6772967
Nature. 1993 Dec 9;366(6455):537-43
8255292
Structure. 1998 May 15;6(5):571-86
9634695
FEMS Microbiol Rev. 2003 Oct;27(4):449-79
14550940
Anal Biochem. 1984 Feb;137(1):266-7
6329026
Arch Microbiol. 2000 May-Jun;173(5-6):325-32
10896210
Arch Microbiol. 1999 May-Jun;171(6):364-70
10369892
J Biol Chem. 1993 Oct 15;268(29):21569-77
8408008
Anal Biochem. 1983 Jul 1;132(1):6-13
6312838
J Bacteriol. 2000 Mar;182(6):1641-9
10692370
Biotechnol Bioeng. 1996 Aug 5;51(3):349-59
18624367
Gene. 1982 Oct;19(3):259-68
6295879
J Bacteriol. 2006 Jul;188(13):4962-9
16788204
Protein Sci. 1997 Mar;6(3):556-68
9070438
Appl Environ Microbiol. 1994 Jul;60(7):2473-82
8074526
J Gen Microbiol. 1992 Jul;138(7):1301-7
1512560
Appl Environ Microbiol. 1998 Jul;64(7):2710-5
9647854
Microbiology. 2003 Jul;149(Pt 7):1771-84
12855729
Arch Microbiol. 2000 Jul-Aug;174(1-2):35-41
10985740
J Biol Chem. 2002 Feb 22;277(8):5858-65
11709550
Appl Environ Microbiol. 2004 Jun;70(6):3253-62
15184119
Appl Environ Microbiol. 2004 Jun;70(6):3222-31
15184115
Appl Environ Microbiol. 2002 Nov;68(11):5265-73
12406713
J Bacteriol. 2002 Jan;184(2):344-9
11751810
Gene. 1995 Dec 1;166(1):175-6
8529885
Gene. 1989 Apr 15;77(1):51-9
2744487
Science. 1997 Jan 24;275(5299):515-8
8999792
J Biol Chem. 2004 Jul 16;279(29):30600-10
15096510
Mol Microbiol. 1991 Feb;5(2):335-42
1904125
Biodegradation. 1990;1(1):19-29
1368139
J Mol Biol. 2000 Feb 18;296(2):701-12
10669618