Structure-Based mutagenesis approaches toward expanding the substrate specificity of d-2-Deoxyribose-5-phosphate aldolase

Abstract

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and d-glyceraldehyde-3-phosphate to generate d-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 Å crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, d-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor™. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT ( Δace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.