Structure and function of the alcohol acyltransferases from yeast and fruit

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Volatile esters are important secondary metabolites that are produced by yeast during fermentation + in plants during fruit ripening.Understanding the biochemistry of the volatile esters is thus of considerable importance in industrial agriculture, winemaking + brewing.Additionally, the enzymes responsible for ester synthesis are now being used in cellular 'factories' to produce fragrances, industrial solvents, fine chemicals + renewable biofuels.However, the detailed structure and function of these enzymes remain poorly understood.This presents a substantial barrier to exploiting such enzymes in food technology and synthetic biology.There are 2 protein families in the industrial brewing yeast Saccharomyces cerevisiae responsible for volatile ester synthesis.Both families function as acyl-CoA: alcohol O-acyltransferases, catalysing ester formation from alcohol + acyl-CoA cosubstrates.We have recently identified 2 model proteins from each family that can be recombinantly expressed + we have begun to study them in vitro.This project will build upon our previous work to characterize the structure + function of these yeast enyzmes + of related acyltransferases from fruit.This is driven by 3 interrelated research questions:What is the basis for acyl-CoA selectivity by the yeast acyltransferases?The two yeast protein families have different activity profiles toward different chain-length acyl-CoAs.We will investigate how this specificity is mediated at the active site. What is the basis for thioesterase activity in the yeast acyltransferases?We have found that the yeast enzymes are promiscuous with regard to alcohol +,unexpectedly, that water can substitute for alcohol during catalysis; thus they can act as acyl-CoA thioesterases. We will determine the mechanistic basis of this activity. Can we extend our methodological toolkit to study fruit acyltransferases?These enzymes, with substantial commercial + ecological significance, have not yet been meaningfully characterised in vitro.All proteins will be recombinantly expressed + purified following established protocols and entered into crystal screens using state-of-the-art robotics.Coupled biochemical assays and GC-MS will be used to study enzyme activity + mechanism.Computational ligand docking will be used to simulate + understand protein-substrate interactions. This project will provide high-level skills training for the student:Molecular biology;recombinant protein expression + purification;protein characterization by biochemical + biophysical methods;enzyme kinetics;site-directed mutagenesis; X-ray crystallography; Protein modeling + ligand docking simulations. This project is directly relevant to Agriculture and Food Security, particularly the areas of Crop Science and Healthy + Safe Food.The expression of alcohol acyltransferase genes correlates directly with crop ripening, + understanding the biochemistry of fruit ripening offers a route towards improving + manipulating fruit flavour + quality via selective breeding or genetic modification.This is thus relevant to several of the goals of these priority areas: Changing food availability, increasing shelf life + increasing the consumer acceptance of fruits to promote a healthier diet. Characterising the acyltransferases from yeast could also influence the production of mass-market consumer beverages that rely upon yeast fermentation, such as beer + wine.Impacts in this arena are thus likely to include reducing cost + ensuring availability.Although less relevant to health themes, yeast-derived fermented beverages are a major industrial concern;the EU alone produces nearly 40bn litres of beer/annum with a sales value of >100bn euros. A further strand was introduced when evident the same techniques could be applied to an uncharacterized membrane protein that is essential in some pathogenic bacteria + thus pursued the first biophysical studies on this novel transport protein to elucidate structure + function.