Novel monooxygenase biocatalysts from the environment and the laboratory

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Methane oxidising bacteria are very important in the environment since they are a key link in the global biogeochemical methane cycle and oxidise methane in many environments such as wetlands, paddy field soils and landfills before this methane is released into the environment. They thereby mitigating the effects of this potent greenhouse gas and reduce global warming. Methane monooxygenase (MMO) is a bacterial enzyme that catalyses the first step in methane oxidation by bacteria. It is of great interest to chemists because it oxidises methane to methanol at ambient temperatures and pressures, a reaction normally requiring high temperatures and pressures and expensive catalysts. MMO is very unusual in that it will also oxidise very many other alkanes, alkenes and aromatic compounds and their substituted derivatives and therefore it has great potential for use as a biocatalyst in biotransformations and bioremediation in 'green chemistry' reactions that are less polluting than traditional chemical routes. The structure of MMO has been the subject of considerable interest for biologists because of this broad substrate specificity and one aim has been to try to understand how the structure of the enzyme allows the catalysis of such a wide range of compounds and how changing its structure by mutagenesis, forced evolution or construction of mutant and hybrid enzymes will alter its catalytic utility. This is an ambitious grant proposal from world experts in the molecular biology and biochemistry of MMO. We propose to construct key mutants in the active site of MMO and to examine the effects on catalysis of key substrates and to manipulate this enzyme in order to be able to define the pathway of entry of substrates into the active site and to generate novel recombinant enzymes which are able to oxidise new substrates. We also aim to define how the different components of the enzyme interact with each other and how the mechanisms of substrate entry and electron transfer pathways to the site of oxidation in MMO are carried out. In a novel approach, we also wish to carry out 'gene mining' from the environment to capture DNA sequences that encode MMO or related di-iron centre monooxygenases in order to be able to construct new and exciting biocatalysts. This will involve the use of a technique called DNA-Stable Isotope Probing (DNA-SIP) which we originally developed in order to be able to define the population structure of active methane oxidising bacteria in the environment. This involves feeding 13C-substrates such as methane to bacteria contained within environmental samples such as soils. Only the active methanotrophs with MMO will be labelled with this heavy stable isotope. We can then isolate the heavy DNA (containing the whole genomes of methanotrophs and related bacteria) encoding MMO and its relatives from all of the DNA from the thousands of non-methanotrophic bacteria present in soil by density gradient centrifugation. By use of the polymerase chain reaction (PCR), we can then isolate novel MMO sequences from previously uncultivated bacteria which can subsequently be stitched into plasmids that we have developed which allow us to recreate novel MMOs with unusual biocatalytic properties. Analysis of these recombinant enzymes will shed light on the mechanism of action of MMO and also generate new and novel biocatalysts with potential for use in industry in non-polluting biotransformation reactions.