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Applied and Environmental Microbiology, November 2002, p. 5265-5273, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5265-5273.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

Thomas J. Smith,{dagger} Susan E. Slade, Nicolas P. Burton,{ddagger} J. Colin Murrell,* and Howard Dalton

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received 8 April 2002/ Accepted 16 July 2002

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an ({alpha}ß{gamma})2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the {alpha} subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44 24 7652 3553. Fax: 44 24 7652 3568. E-mail: cmurrell{at}bio.warwick.ac.uk.

{dagger} Present address: Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB, United Kingdom.

{ddagger} Present address: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

Applied and Environmental Microbiology, November 2002, p. 5265-5273, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5265-5273.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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