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Applied and Environmental Microbiology, October 2007, p. 6460-6467, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00823-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Mutagenesis of the "Leucine Gate" To Explore the Basis of Catalytic Versatility in Soluble Methane Monooxygenase{triangledown}

Elena Borodina,1,{dagger} Tim Nichol,2,{dagger} Marc G. Dumont,1 Thomas J. Smith,2* and J. Colin Murrell1

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom,1 Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom2

Received 11 April 2007/ Accepted 7 August 2007

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.

* Corresponding author. Mailing address: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, United Kingdom. Phone: 44 114 225 3042. Fax: 44 114 225 3066. E-mail: t.j.smith{at}shu.ac.uk

{triangledown} Published ahead of print on 17 August 2007.

{dagger} E. Borodina and T. Nichol made equal contributions to this work.

Applied and Environmental Microbiology, October 2007, p. 6460-6467, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.00823-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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