Structurally informed mutagenesis of a stereochemically promiscuous aldolase produces mutants that catalyze the diastereoselective syntheses of all four stereoisomers of 3-deoxy-hexulosonic acid

DOI: 10.1021/acscatal.2c03285

Authors: Sylvain F. Royer (University of Bath) , Xuan Gao (University of Bristol) , Robin R. Groleau (University of Bath) , Marc W. Van Der Kamp (University of Bristol) , Steven D. Bull (University of Bath) , Michael J. Danson (University of Bath) , Susan J. Crennell (University of Bath)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: September 2022
Diamond Proposal Number(s): 1226

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.

Journal Keywords: enzyme engineering; stereospecificity; aldolase; crystal structures; Sulfolobus solfataricus; carbon−carbon bond formation

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 08/09/2022 09:08

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)