Rational design of stereoselectivity in the class II pyruvate aldolase BphI

BphI, a pyruvate-specific class II aldolase, catalyzes the reversible carbon-carbon bond formation of 4-hydroxy-2-oxoacids up to eight carbons in length. During the aldol addition catalyzed by BphI, the S-configured stereogenic center at C4 is created via attack of a pyruvate enolate intermediate on the si face of the aldehyde carbonyl of acetaldehyde to form 4(S)-hydroxy-2-oxopentanoate. Replacement of a Leu-87 residue within the active site of the enzyme with polar asparagine and bulky tryptophan led to enzymes with no detectable aldolase activity. These variants retained decarboxylase activity for the smaller oxaloacetate substrate, which is not inhibited by excess 4-hydroxy-2-oxopentanoate, confirming the results from molecular modeling that Leu-87 interacts with the C4-methyl of 4(S)-hydroxy-2-oxoacids. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids by relieving the steric hindrance between the 5-methyl group of these compounds and the hydroxyl substituent on the phenyl ring of Tyr-290. The resultant enzymes were shown to exclusively utilize only 4(R)- and not 4(S)-hydroxy-2-oxopentanoate as the substrate. Polarimetric analysis confirmed that the double variants are able to synthesize 4-hydroxy-2-oxoacids up to eight carbons in length, which were the opposite stereoisomer compared to those produced by the wild-type enzyme. Overall the k(cat)/K(m) values for pyruvate and aldehydes in the aldol addition reactions were affected ≤10-fold in the double variants relative to the wild-type enzyme. Thus, stereocomplementary class II pyruvate aldolases are now available to create chiral 4-hydroxy-2-oxoacid skeletons as synthons for organic reactions.