SMITH, T. J., SLADE, S. E., BURTON, N. P., MURRELL, J. C. and DALTON, H. (2002). Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase. Applied and Environmental Microbiology, 68 (11), 5265-5273.Full text not available from this repository.
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 an active form of the hydroxylase component, which is an (alpha beta gamma)2 complex containing the binuclear iron active site. We previously reported an homologous expression system that enabled expression of recombinant wild-type sMMO in a derivative of Ms. trichosporium OB3b in which the chromosomal copy of the sMMO-encoding operon had been partially deleted. Here we report substantial development of this methodology to produce a system for 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 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% of wild-type activity toward toluene but only 17% wild-type activity toward propene, so its substrate profile was significantly altered. Thus we have demonstrated proof of principle for protein engineering of this uniquely versatile enzyme.
|Research Institute, Centre or Group:||Biomedical Research Centre|
|Depositing User:||Ann Betterton|
|Date Deposited:||19 Feb 2008|
|Last Modified:||09 Dec 2009 18:23|
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