Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.
Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.
Wada
Masaru
M
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Hsu
Che-Chang
CC
Franke
Dirk
D
Mitchell
Michael
M
Heine
Andreas
A
Wilson
Ian
I
Wong
Chi-Huey
CH
D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism
D000447:Aldehydes; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase