Mutagenic studies into the catalytic versatility of soluble methane monooxygenase

NICHOL, Tim (2011). Mutagenic studies into the catalytic versatility of soluble methane monooxygenase. Doctoral, Sheffield Hallam University.

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Abstract

Soluble methane monooxygenase (sMMO) is a multicomponent bacterial enzyme that catalyzes the oxidation of methane to methanol, as well as oxidizing many other adventitious substrates. A number of mutagenic studies were carried out on the sMMO enzyme of Methylosinus trichosporium OB3b in order to gain insight into sMMO and probe how structural aspects relate to function of the enzyme. Leu110 within the hydroxylase a-subunit of sMMO has been proposed as a possible gating residue, controlling access of substrate to the active site (Rosenzweig et al. 1997). A range of site directed mutants were created at the 110 position and screened for activity with a number of aromatic substrates. All mutants showed relaxed regioselectivity with all substrates assayed. However no evidence of a gating residue was found, indicating that Leu110 is more important in determining regioselectivity than substrate access to the active site. Comparison to the highly similar butane monooxygenase led to the creation of three site directed mutants: M184V F282L and C151T. M184V and C151T showed small changes in regioselectivity and reduced activity with most substrates. The M184V mutant showed relaxed regioselectivity and a novel oxidation product with the substrate mesitylene which may have implications for substrate trafficking. The F282L mutant produced a stable enzyme which had no activity with any of the substrates tested, showing Phe282 is important for the enzyme function. A random mutagenesis experiment was devised and a colorimetric screen for the oxidation of triaromatic compounds was used to screen mutant libraries for activity towards anthracene and phenanthrene. However no activity towards triaromatic compounds was detected. In order to improve the cloning strategies and to make creation of mutant libraries easier, a novel expression vector pT2ML was created. The pT2ML vector reduces the number of cloning steps required to make soluble methane monooxygenase mutants. This expression system was used to make a site directed mutants F188Aand N116G in order to complement previous site directed mutant studies, as well as a recombinant wild type mutant in order to asses the activity of the new expression system which is comparible to the wild type enzyme.

Item Type:	Thesis (Doctoral)
Contributors:	Thesis advisor - Smith, Thomas [0000-0002-4246-5020]
Additional Information:	Proquest number 30994 SHU Thesis no. 26949
Research Institute, Centre or Group - Does NOT include content added after October 2018:	Sheffield Hallam Doctoral Theses
Depositing User:	Jill Hazard
Date Deposited:	09 Aug 2017 12:21
Last Modified:	03 May 2023 02:05
URI:	https://shura.shu.ac.uk/id/eprint/16489

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