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

2 Citations (SciVal)

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 Sept 2022
DOIs
Publication statusPublished - 16 Sept 2022

Bibliographical note

We thank the BBSRC (S.F.R.; M.W.v.d.K, BB/M026280/1) and EPSRC CDT in Catalysis (R.R.G., EP/L016443/1) for funding. We acknowledge the valuable contributions made to this project by many undergraduate and masters project students: Luke Haslett, Emily Gostling, Marina Angelopoulou, Laurence Devesse, Leigh Knight, Marina Vabistsevits, Jennifer Frewin, Pooja Jhugroo, and Jennifer Gurney. The authors would like to thank Diamond Light Source for beamtime (proposal mx1226) and the staff of beamlines I03 and I04 for assistance with data collection. Characterization facilities were provided by the Material and Chemical Characterization Facility (MC (2)) at the University of Bath (https://doi.org/10.15125/mx6j-3r54), and we wish to thank Dr. Kathryn Proctor for her help and expertise. MD simulations were conducted using the computational facilities of the Advanced Computing Research Centre, University of Bristol.

Keywords

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

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry

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