PMID:15967977
| Citation |
Hsu, CC, Hong, Z, Wada, M, Franke, D and Wong, CH (2005) Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase. Proc. Natl. Acad. Sci. U.S.A. 102:9122-6 |
|---|---|
| Abstract |
An efficient L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase was created by directed evolution from the Escherichia coli D-Neu5Ac (N-acetylneuraminic acid, D-sialic acid) aldolase. Five rounds of error-prone PCR and iterative screening were performed with sampling of 10(3) colonies per round. The specificity constant (kcat/Km) of the unnatural sugar L-KDO is improved to a level equivalent to the wild-type D-sialic acid aldolase for its natural substrate, D-Neu5Ac. The final evolved enzyme exhibits a >1,000-fold improved ratio of the specificity constant [kcat/Km (L-KDO)]/[kcat/Km (D-sialic acid)]. The protein sequence of the evolved aldolase showed eight amino acid changes from the native enzyme, with all of the observed changes occurring outside of the active site. Our effort demonstrates that an enzyme can be rapidly altered to accept enantiomeric substrates with screening of a small population of colonies iteratively toward the target substrate with improved catalytic efficiency. This work provides a method for the synthesis of enantiomeric sugars and for the study of enantiomeric catalysis affected by remote mutations. |
| Links |
PubMed PMC1166642 Online version:10.1073/pnas.0504033102 |
| Keywords |
Amino Acids/chemistry; Binding Sites; Carbohydrate Sequence; Catalysis; Cell Membrane/metabolism; Crystallography, X-Ray; Escherichia coli/metabolism; Evolution, Molecular; Fructose-Bisphosphate Aldolase/chemistry; Kinetics; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Mutagenesis; Mutation; N-Acetylneuraminic Acid/analogs & derivatives; N-Acetylneuraminic Acid/chemistry; Oxo-Acid-Lyases/chemistry; Oxo-Acid-Lyases/genetics; Peptides/chemistry; Polymerase Chain Reaction; Protein Conformation; Protein Engineering; Protein Structure, Secondary; Substrate Specificity; Sugar Acids/chemistry |
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