Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens PIV-1
Quaternary carbon containing compounds exist in natural and fossil oil-derived products and are used in many chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment, and to date, their biodegradation by microorganisms has remained largely unknown. Here, we investigated the degradation of the quaternary carbon compound containing model compound pivalate (2,2,-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens PIV-1. Whole cell coenzyme A (CoA) ester pool analyses, along with in vitro enzyme activity measurements and mass spectrometric analysis of intermediates indicated a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed via intermediates of the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA intermediate and the cleavage of 3-methly-3-hydroxyglutaryl-CoA to acetyl-CoA to acetoacetate. We provide the complete genome of Thauera humireducens PIV-1 that empowered the proteome analyses based identification of pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. The results provide first experimental insights in the genes and enzymes involved in the biodegradation of a quaternary carbon containing model compound, which serves as a blueprint for the degradation of related quaternary carbon containing compounds.