Mutagenesis of the "Leucine Gate" to explore the basis of catalytic versatility in soluble methane monooxygenase

BORODINA, E., NICHOL, T., DUMONT, M. G., SMITH, T. J. and MURRELL, J. C. (2007). Mutagenesis of the "Leucine Gate" to explore the basis of catalytic versatility in soluble methane monooxygenase. Applied and environmental microbiology, 73 (20), 6460-6467.

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      Link to published version:: 10.1128/AEM.00823-07

      Abstract

      Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent non-heme oxygenase that naturally oxidizes methane to methanol and can also co-oxidize a wide range of adventitious substrates, including mono- and di-aromatic hydrocarbons. Leucine 110, at the mouth of the active site in the ά-subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control 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 in 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 mutants at position 110 in the alpha-subunit of sMMO. All the mutants (with Gly, Cys, Arg and Tyr, respectively, at this position) showed a relaxation of the wild-type regioselectivity 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 at position 110 also showed a shift in regioselectivity during naphthalene hydroxylation from the 2- to the 1-position. No evidence was found, however, that mutagenesis of Leu 110 could allow very large substrates to enter the active site, since the mutants (like the wild-type) were inactive towards 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 size of substrates that can enter the active site.

      Item Type: Article
      Additional Information: © American Society for Microbiology published in Applied and Environmental Microbiology, October 2007,73(20), 6460-6467
      Research Institute, Centre or Group: Biomedical Research Centre
      Identification Number: 10.1128/AEM.00823-07
      Depositing User: Ann Betterton
      Date Deposited: 09 Nov 2007
      Last Modified: 21 Dec 2010 11:30
      URI: http://shura.shu.ac.uk/id/eprint/365

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