Dataset Information

Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.

ABSTRACT: Soluble methane monooxygenase (sMMO) from methane-oxidising bacteria can oxygenate more than 100 hydrocarbons and is one of the most catalytically versatile biological oxidation catalysts. Expression of recombinant sMMO has to date not been achieved in Escherichia coli and so an alternative expression system must be used to manipulate it genetically. Here we report substantial improvements to the previously described system for mutagenesis of sMMO and expression of recombinant enzymes in a methanotroph (Methylosinus trichosporium OB3b) expression system. This system has been utilised to make a number of new mutants and to engineer sMMO to increase its catalytic precision with a specific substrate whilst increasing activity by up to 6-fold. These results are the first 'proof-of-principle' experiments illustrating the feasibility of developing sMMO-derived catalysts for diverse applications.

SUBMITTER: Lock M

PROVIDER: S-EPMC5812538 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.

Lock Malcolm M Nichol Tim T Murrell J Colin JC Smith Thomas J TJ

FEMS microbiology letters 20170701 13

Soluble methane monooxygenase (sMMO) from methane-oxidising bacteria can oxygenate more than 100 hydrocarbons and is one of the most catalytically versatile biological oxidation catalysts. Expression of recombinant sMMO has to date not been achieved in Escherichia coli and so an alternative expression system must be used to manipulate it genetically. Here we report substantial improvements to the previously described system for mutagenesis of sMMO and expression of recombinant enzymes in a metha ...[more]

PMID: 28854685

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Project description:The controlled oxidation of methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, methane monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O(2). The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent monooxygenases can be measured. Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical environments. P* converts to a second peroxodiiron(III) unit, H(peroxo), in a process accompanied by the transfer of a proton, probably with the assistance of a residue near the active site. Proton-promoted O-O bond scission and rearrangement of the diiron core then leads to a diiron(IV) unit, termed Q, that is directly responsible for the oxidation of methane to methanol. In one section of this Account, we provide a detailed discussion of these processes, with particular emphasis on possible structures of the intermediates. The geometries of P* and H(peroxo) are currently unknown, and recent synthetic modeling chemistry has highlighted the need for further structural characterization of Q, currently assigned as a di(μ-oxo)diiron(IV) "diamond core." In another section of the Account, we discuss in detail proton transfer during the O(2) activation events. The role of protons in promoting O-O bond cleavage, thereby initiating the conversion of H(peroxo) to Q, was previously a controversial topic. Recent studies of the mechanism, covering a range of pH values and in D(2)O instead of H(2)O, confirmed conclusively that the transfer of protons, possibly at or near the active site, is necessary for both P*-to-H(peroxo) and H(peroxo)-to-Q conversions. Specific mechanistic insights into these processes are provided. In the final section of the Account, we present our view of experiments that need to be done to further define crucial aspects of sMMO chemistry. Here our goal is to detail the challenges that we and others face in this research, particularly with respect to some long-standing questions about the system, as well as approaches that might be used to solve them.

Dataset Information

Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.

Publications

Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.

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