Document Detail


Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase.
MedLine Citation:
PMID:  15967977     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Che-Chang Hsu; Zhangyong Hong; Masaru Wada; Dirk Franke; Chi-Huey Wong
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-06-20
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  102     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2005 Jun 
Date Detail:
Created Date:  2005-06-29     Completed Date:  2005-08-11     Revised Date:  2013-06-09    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  9122-6     Citation Subset:  IM    
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.
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MeSH Terms
Descriptor/Qualifier:
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*,  chemistry
Oxo-Acid-Lyases / chemistry,  genetics*
Peptides / chemistry
Polymerase Chain Reaction
Protein Conformation
Protein Engineering
Protein Structure, Secondary
Substrate Specificity
Sugar Acids / chemistry*
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Peptides; 0/Sugar Acids; 1069-03-0/2-keto-3-deoxyoctonate; 131-48-6/N-Acetylneuraminic Acid; 24967-27-9/2-deoxy-2,3-dehydro-N-acetylneuraminic acid; EC 4.1.2.13/Fructose-Bisphosphate Aldolase; EC 4.1.3.-/Oxo-Acid-Lyases; EC 4.1.3.3/N-acetylneuraminate lyase
Comments/Corrections

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