Structurally informed site-directed mutagenesis of a stereochemically promiscuous aldolase to afford stereochemically complementary biocatalysts.
2-Keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus is a highly thermostable type I aldolase that can catalyze carbon-carbon bond formation using nonphosphorylated substrates. However, it exhibits poor diastereocontrol in many of its aldol reactions, including the reaction of its natural substrates, pyruvate and D-glyceraldehyde, which afford a 55:45 mixture of D-2-keto-3-deoxygluconate (D-KDGlu) and D-2-keto-3-deoxy-galactonate (D-KDGal). We have employed detailed X-ray crystallographic structural information of this aldolase bound to these diastereoisomeric aldol products to selectively target specific amino acids for mutation for the rapid creation of stereochemically complementary mutants that catalyze either (Re)- or (Si)-facial selective aldol reactions to afford either D-KDGlu or D-KDGal with good levels of diastereocontrol.
Royer
Sylvain F
SF
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom BA2 7AY.
Haslett
Luke
L
Crennell
Susan J
SJ
Hough
David W
DW
Danson
Michael J
MJ
Bull
Steven D
SD
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013237:Stereoisomerism; D048229:Sulfolobus solfataricus / Q000201:enzymology
C021470:2-keto-3-deoxygluconate aldolase; D000446:Aldehyde-Lyases