Structurally Informed Mutagenesis of a Stereochemically Promiscuous Aldolase Produces Mutants That Catalyze the Diastereoselective Syntheses of All Four Stereoisomers of 3-Deoxy-hexulosonic Acid.

ABSTRACT: A 2-keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus catalyzes the nonstereoselective aldol reaction of pyruvate and d-glyceraldehyde to produce 2-keto-3-deoxygluconate (d-KDGlc) and 2-keto-3-deoxy-d-galactonate (d-KDGal). Previous investigations into curing the stereochemical promiscuity of this hyperstable aldolase used high-resolution structures of the aldolase bound to d-KDGlc or d-KDGal to identify critical amino acids involved in substrate binding for mutation. This structure-guided approach enabled mutant variants to be created that could stereoselectively catalyze the aldol reaction of pyruvate and natural d-glyceraldehyde to selectively afford d-KDGlc or d-KDGal. Here we describe the creation of two further mutants of this Sulfolobus aldolase that can be used to catalyze aldol reactions between pyruvate and non-natural l-glyceraldehyde to enable the diastereoselective synthesis of l-KDGlc and l-KDGal. High-resolution crystal structures of all four variant aldolases have been determined (both unliganded and liganded), including Variant 1 with d-KDGlc, Variant 2 with pyruvate, Variant 3 with l-KDGlc, and Variant 4 with l-KDGal. These structures have enabled us to rationalize the observed changes in diastereoselectivities in these variant-catalyzed aldol reactions at a molecular level. Interestingly, the active site of Variant 4 was found to be sufficiently flexible to enable catalytically important amino acids to be replaced while still retaining sufficient enzymic activity to enable production of l-KDGal.

SUBMITTER: Royer SF 

PROVIDER: S-EPMC9486944 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature