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

• Published in: ACS catalysis Vol. 12; no. 18; pp. 11444 - 11455
• Main Authors: Royer, Sylvain F., Gao, Xuan, Groleau, Robin R., van der Kamp, Marc W., Bull, Steven D., Danson, Michael J., Crennell, Susan J.
• Format: Journal Article
• Language: English
• Published: American Chemical Society 16.09.2022
• Subjects: enzyme engineering; aldolase; Sulfolobus solfataricus; stereospecificity; crystal structures; carbon−carbon bond formation
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.2c03285

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.