Document Detail


The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity.
MedLine Citation:
PMID:  18392864     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
CYP102A1 is a highly active water-soluble bacterial monooxygenase that contains both substrate-binding heme and diflavin reductase subunits, all in a single polypeptide that has been called a "self-sufficient enzyme." Several years ago we developed a procedure called "scanning chimeragenesis," where we focused on residues 73-82 of CYP102A1, which contact approximately 40% of the substrates palmitoleic acid and N-palmitoylglycine [Murataliev et al. (2004) Biochemistry 43:1771-1780]. These residues were replaced with the homologous residues of CYP4C7. In the current work, that study has been expanded to include residue 87. Phenylalanine 87 of wild-type CYP102A1 was replaced with the homologous residue of CYP4C7, leucine, as well as with alanine. The full-sized chimeric proteins C(73-78, F87L), C(73-78, F87A), C(75-80, F87L), C(75-80, F87A), C(78-82, F87L) and C(78-82, F87A) have been purified and characterized. Wild-type CYP102A1 is most active toward fatty acids (both lauric and palmitic acids produce omega-1, omega-2, and omega-3 hydroxylated fatty acids), but it also catalyzes the oxidation of farnesol to three products (2, 3- and 10,11-epoxyfarnesols and 9-hydroxyfarnesol). All of the F87-mutant chimeric proteins show dramatic decreases in activities with the natural CYP102A1 substrates. In contrast, C(78-82, F87A) and C(78-82, F87L) have markedly increased activities with farnesol, with the latter showing a 5.7-fold increase in catalytic activity as compared to wild-type CYP102A1. C(78-82, F87L) produces 10,11-epoxyfarnesol as the single primary metabolite. The results show that chimeragenesis involving only the second half of SRS-1 plus F87 is sufficient to change the substrate selectivity of CYP102A1 from fatty acids to farnesol and to produce a single primary product.
Authors:
Chiung-Kuang J Chen; Tatiana Kh Shokhireva; Robert E Berry; Hongjun Zhang; F Ann Walker
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-04-08
Journal Detail:
Title:  Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry     Volume:  13     ISSN:  0949-8257     ISO Abbreviation:  J. Biol. Inorg. Chem.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-05-29     Completed Date:  2008-09-30     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9616326     Medline TA:  J Biol Inorg Chem     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  813-24     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of Arizona, Tucson, AZ, 85721-0041, USA.
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MeSH Terms
Descriptor/Qualifier:
Bacterial Proteins / chemistry*,  genetics*
Catalysis
Cytochrome P-450 Enzyme System / chemistry*,  genetics*
DNA Primers
Farnesol / metabolism
Lauric Acids / metabolism
Magnetic Resonance Spectroscopy
Models, Molecular
Mutant Chimeric Proteins / chemistry*,  genetics*
NADP / metabolism
NADPH-Ferrihemoprotein Reductase / chemistry*,  genetics*
Oxidation-Reduction
Palmitic Acid / metabolism
Point Mutation / physiology*
Protein Conformation
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spectrophotometry, Atomic
Spectrophotometry, Ultraviolet
Substrate Specificity
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/DNA Primers; 0/Lauric Acids; 0/Mutant Chimeric Proteins; 143-07-7/lauric acid; 4602-84-0/Farnesol; 53-59-8/NADP; 57-10-3/Palmitic Acid; 9035-51-2/Cytochrome P-450 Enzyme System; 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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