Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars.
BACKGROUND
Exploitation and improvement of enzymes as catalysts for organic synthesis is of current interest in biocatalysis. A representative enzyme for investigation is the Escherichia coli D-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes the highly specific reversible aldol reaction using the D-configurated KDPG as substrate.


RESULTS
Using in vitro evolution, the aldolase has been converted into aldolases with improved catalytic efficiency, altered substrate specificity and stereoselectivity. In particular, some evolved aldolases capable of accepting both D- and L- glyceraldehyde in the non-phosphorylated form as substrates for reversible aldol reaction have been obtained, providing a new direction to the enzymatic synthesis of both D- and L-sugars.


CONCLUSIONS
This research has demonstrated the effectiveness of using in vitro evolution to rapidly alter the properties of an aldolase to improve its utility in asymmetric synthesis. The evolved aldolases, differing from the native enzyme which is highly phosphate- and D-sugar-dependent, catalyze the efficient synthesis of both D- and L-sugars from non-phosphorylated aldehydes and pyruvate. The principles and strategies described in this study should be applicable to other aldolases to further expand the scope of their synthetic utility.
Fong
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Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Machajewski
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Wong
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D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002241:Carbohydrates / Q000096:biosynthesis* / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D002241:Carbohydrates; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase