Novel monooxygenase biocatalysts from the environment and the laboratory

甲烷氧化细菌在环境中非常重要，因为它们是全球地球化学甲烷循环中的关键环节，并且在甲烷释放到环境中之前在许多环境如湿地、稻田土壤和垃圾填埋场中氧化甲烷。它们从而减轻了这种强效温室气体的影响，减少了全球变暖。甲烷单加氧酶（MMO）是一种细菌酶，催化细菌氧化甲烷的第一步。化学家对它非常感兴趣，因为它在环境温度和压力下将甲烷氧化为甲醇，该反应通常需要高温和高压以及昂贵的催化剂。MMO是非常不寻常的，因为它也将氧化非常多的其它烷烃、烯烃和芳香族化合物及其取代衍生物，因此它具有在“绿色化学”反应中用作生物转化和生物修复中的生物催化剂的巨大潜力，所述反应比传统化学途径污染更少。MMO的结构一直是生物学家相当感兴趣的主题，因为这种广泛的底物特异性，一个目标是试图了解酶的结构如何允许催化如此广泛的化合物，以及如何通过诱变、强制进化或构建突变体和杂合酶来改变其结构将改变其催化效用。这是一个雄心勃勃的资助计划，来自世界各地的MMO分子生物学和生物化学专家。我们建议在MMO的活性位点构建关键突变体，并研究关键底物对催化的影响，并操纵这种酶，以便能够确定底物进入活性位点的途径，并产生能够氧化新底物的新型重组酶。我们还旨在确定酶的不同组分如何相互作用，以及如何在MMO中进行底物进入和电子转移途径到氧化位点的机制。在一种新的方法中，我们还希望从环境中进行“基因挖掘”，以捕获编码MMO或相关二铁中心单加氧酶的DNA序列，以便能够构建新的和令人兴奋的生物催化剂。这将涉及使用一种称为DNA稳定同位素探测（DNA-SIP）的技术，我们最初开发该技术是为了能够确定环境中活性甲烷氧化细菌的种群结构。这涉及将甲烷等13 C底物喂给土壤等环境样品中所含的细菌。只有具有MMO的活性甲烷氧化菌将用这种重稳定同位素标记。然后，我们可以通过密度梯度离心从土壤中存在的数千种非甲烷氧化细菌的所有DNA中分离编码MMO及其亲属的重DNA（包含甲烷氧化菌和相关细菌的整个基因组）。通过使用聚合酶链反应（PCR），我们可以从以前未培养的细菌中分离出新的MMO序列，然后将其缝合到我们开发的质粒中，从而使我们能够重新创建具有不寻常生物催化特性的新型MMO。对这些重组酶的分析将揭示MMO的作用机制，并产生新的和新颖的生物催化剂，具有在工业上用于无污染的生物转化反应的潜力。

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.