Document Detail


Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.
MedLine Citation:
PMID:  10839169     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.
Authors:
Q S Li; U Schwaneberg; P Fischer; R D Schmid
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Chemistry (Weinheim an der Bergstrasse, Germany)     Volume:  6     ISSN:  0947-6539     ISO Abbreviation:  Chemistry     Publication Date:  2000 May 
Date Detail:
Created Date:  2000-06-15     Completed Date:  2000-06-15     Revised Date:  2009-08-04    
Medline Journal Info:
Nlm Unique ID:  9513783     Medline TA:  Chemistry     Country:  GERMANY    
Other Details:
Languages:  eng     Pagination:  1531-6     Citation Subset:  IM    
Affiliation:
Institut für Technische Biochemie, Universität Stuttgart, Germany. itbrsc@po.uni-stuttgart.de
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MeSH Terms
Descriptor/Qualifier:
Bacillus megaterium / enzymology*
Bacterial Proteins*
Cytochrome P-450 Enzyme System / genetics*,  metabolism
Fatty Acids / metabolism
Hydroxylation
Indoles / metabolism*
Kinetics
Mixed Function Oxygenases / genetics*,  metabolism
Mutagenesis, Site-Directed
NADPH-Ferrihemoprotein Reductase
Substrate Specificity
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/Fatty Acids; 0/Indoles; 120-72-9/indole; 479-41-4/indirubin; 9035-51-2/Cytochrome P-450 Enzyme System; EC 1.-/Mixed Function Oxygenases; EC 1.6.2.4/NADPH-Ferrihemoprotein Reductase; EC 1.6.2.4/flavocytochrome P450 BM3 monoxygenases

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


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