title: Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase.
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.
authors.affiliation: Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Che-Chang
authors.initials: CC
authors.lastname: Hsu
authors.forename: Zhangyong
authors.initials: Z
authors.lastname: Hong
authors.forename: Masaru
authors.initials: M
authors.lastname: Wada
authors.forename: Dirk
authors.initials: D
authors.lastname: Franke
authors.forename: Chi-Huey
authors.initials: CH
authors.lastname: Wong
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013400:Sugar Acids / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D010455:Peptides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D005634:Fructose-Bisphosphate Aldolase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D019158:N-Acetylneuraminic Acid