Livestock produce large amounts of methane as part of their normal digestive process. Methane emissions represent a significant source of greenhouse gases, particularly in New Zealand. They not only contribute to global climate change, but also cause the energy loss of host animals. There are a number of ways to reduce methane emissions that are currently being investigated worldwide. The principal methane forming organisms in the rumen are methanogenic archaea. Many of their enzymes are evolutionarily and biochemically distinct from those of bacteria, humans and ruminants thus they could be specifically targeted using small molecules as inhibitors.
We are exploring novel non-toxic and environmentally-friendly inhibitors of rumen methanogens to reduce methane emissions using a pipeline for cloning, expression, structure determination, enzyme assay development, rumen culture and in vitro testing. The developed assays and enzyme structures are being utilised to perform high-throughput screening. One example of this approach is high-throughput screening of methanogen inhibitors using the enzyme target L-sulfolactate dehydrogenase (ComC, EC 1.1.1.272). It is a NADH/NADPH-dependent oxido-reductase that catalyses the inter-conversion of 2-hydroxyacids into their corresponding 2-oxoacids. This is an essential process involved in coenzyme M synthesis for the methanogenesis pathway in archaea. Recombinant ComC from Methanobrevibacter sp. SM9 has been expressed in Escherichia coli and purified by ion metal affinity chromatography. Enzyme activity was observed after a pretreatment with 0.3 mM NADH. The reverse reaction of ComC, i.e. consumption of NADH, was monitored using oxaloacetate as the preferred substrate at the optimal pH 7.5 and ionic strength 100 mM KCl. The kinetic parameters were: KM (NADH) 148 µM and KM (oxaloacetate) 51.4 µM. The ComC assay has been used to screen compounds libraries which has resulted in the discovery of potential inhibitors that will be tested in animal trials.