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

Sylvain F. Royer, Xuan Gao, Robin Groleau, Marc W. van der Kamp, Steven Bull, Michael J. Danson, Susan Crennell

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)11444-11455
Number of pages12
JournalACS Catalysis
Volume12
Issue number18
Early online date6 Sep 2022
DOIs
Publication statusPublished - 16 Sep 2022

Keywords

  • Sulfolobus solfataricus
  • aldolase
  • carbon-carbon bond formation
  • crystal structures
  • enzyme engineering
  • stereospecificity

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

Fingerprint

Dive into the research topics of 'Structurally Informed Mutagenesis of a Stereochemically Promiscuous Aldolase Produces Mutants That Catalyze the Diastereoselective Syntheses of All Four Stereoisomers of 3Deoxy-hexulosonic Acid'. Together they form a unique fingerprint.

Cite this