Document Detail


Design and properties of human D-amino acid oxidase with covalently attached flavin.
MedLine Citation:
PMID:  10716694     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.
Authors:
A A Raibekas; K Fukui; V Massey
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  97     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2000 Mar 
Date Detail:
Created Date:  2000-04-24     Completed Date:  2000-04-24     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  3089-93     Citation Subset:  IM    
Affiliation:
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Benzoates / metabolism
Catalysis
D-Amino-Acid Oxidase / chemistry,  genetics,  isolation & purification,  metabolism*
DNA Primers
Flavins / metabolism*
Humans
Hydrolysis
Kinetics
Mutagenesis, Site-Directed
Oxidation-Reduction
Spectrum Analysis
Grant Support
ID/Acronym/Agency:
GM 11106/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Benzoates; 0/DNA Primers; 0/Flavins; EC 1.4.3.3/D-Amino-Acid Oxidase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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