Evolving a stereochemically promiscuous aldolase to create new classes of stereoselective biocatalyst for asymmetric synthesis

This proposal describes the use of a thermostable enzyme to prepare new types of chiral compounds as building blocks for synthesising important intermediates for the pharmaceutical and fine chemical industry. The scope and limiation of the enzyme to catalyse a specific reaction with a wide range of substrates will be investigated. The enzyme contains an active site where catalysis takes place, and its structure has been characterised at a molecular level by X-ray crystallography. Knowledge of the structure of this active site will enable us to generate new 'mutant' catalysts with their own specificity profiles as improved biocatalysts for synthesis. These mutation studies will fall into two categories: firstly, we will deliberately introduce specific changes into the enzyme; and secondly, we will generate random mutations to afford large libraries of catalysts. Screening protocols will be developed that will enable us to rapidly identify mutants with useful catalytic activity, with the aim of identifying biocatalysts that carry out reactions not currently observed in nature. Concurrently, we will also develop a chemical approach to the synthesis of these type of compounds, using small chiral molecules as alternative catalysts. A number of compounds produced in this study will then be converted into novel highly fuctionalised intermediates of use for the synthesis of biologically-important natural products.

Aldolases are powerful biocatalysts for stereoselective carbon-carbon bond-forming reactions, and can be used to prepare enantiopure aldol products containing two new stereocentres in high diastereomeric excess. They are used for the asymmetric synthesis of complex carbohydrates, but their use is restricted by a narrow specificity profile for phosphorylated aldehyde substrates. We have characterised a highly-thermostable aldolase from Sulfolobus solfataricus that exhibits a broad specificity for the aldol reaction of pyruvate with a wide range of non-phosphorylated aldehydes; thus it is potentially a highly-versatile biocatalyst for asymmetric synthesis. However, it catalyses the aldol reaction of pyruvate with D-glyceraldehyde with a remarkable lack of stereocontrol, affording a 50:50 diastereoisomeric mixture of D-2-keto-3-deoxygluconate and D-2-keto-3-deoxygalactonate in excellent yield. It also catalyses the aldol reaction of L-glyceraldehyde with pyruvate to give a 50:50 mixture of the opposing diastereoisomers. We have determined high-resolution structures of the aldolase with a variety of ligands bound, and so have a detailed picture of the structural basis of the enzyme's promiscuity. We will use this substrate promiscuity to our advantage. That is, we are in an excellent position to generate, using a variety of mutagenic strategies, a series of aldolases of defined specificities, both of those currently possessed by the native enzyme, but also of a novel type not currently observed in Nature. We will use site-directed mutagenesis, directed evolution and substrate engineering, coupled with novel screening strategies, to achieve this goal. Concurrently, we will also develop a chemical approach to synthesise these types of compounds using small chiral molecules as alternative catalysts. A number of compounds produced in this study will be converted into novel highly-fuctionalised intermediates for the synthesis of biologically-important products.