Structural and mechanistic analysis of carbon-carbon bond hydrolases and lyases for potential industrial applications

The formation or cleavage of carbon-carbon bonds is often thermodynamically unfavourable and, using abiotic catalysts, only achieved under harsh reaction conditions. As an alternative, numerous enzymes are able to catalyse equivalent reactions and constitute versatile biocatalytic tools for the production of high value chemical building blocks. Three such catalysts have now been analysed with respect to their structure, function, reaction mechanism and/or substrate specificity. The Bacillus subtilis phenolic acid decarboxylase (BsPAD) is a carbon-carbon bond lyase. Despite the availability of an apo structure, the enzyme’s reaction mechanism remained speculative. Within this work, an active site mutant library was prepared and analysed and the structure of one of the variants could be determined in complex with its phenolic acid substrate. Using combined structural and kinetic data, a catalytic mechanism was proposed and confirmed previous modelling approaches. In addition, the codon optimised genes of two carbon-carbon bond hydrolases were subcloned for recombinant protein production. Phloretin hydrolase (Phy), a retro-Friedel Crafts hydrolase from Eubacterium ramulus, was purified and crystallised. Although the crystals’ diffraction quality was too poor for structure determination, a fluorescence spectrum revealed Phy to bind zinc. Together with a substrate screen and kinetic data from an active site mutant library, these findings gave novel insights into a unique metal dependent hydrolase. As a member of the β-diketone cleaving family of enzymes, the oxidised polyvinyl alcohol hydrolase (OPH) from Pseudomonas sp. VM15C was analysed. After extensive solubility screening and construct optimisation, OPH could, for the first time, be purified as a soluble fusion construct with an N-terminal GST tag. The fused protein crystallised and now awaits further structural and mechanistic analysis. It is hoped that the data obtained as part of this project will now advance the understanding of the characterised biocatalysts and create a basis for their optimisation and use in industrial applications