Document Detail


Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.
MedLine Citation:
PMID:  10415125     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.
Authors:
J Wang; E Pichersky
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  368     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  1999 Aug 
Date Detail:
Created Date:  1999-09-08     Completed Date:  1999-09-08     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  172-80     Citation Subset:  IM    
Copyright Information:
Copyright 1999 Academic Press.
Affiliation:
Biology Department, University of Michigan, Ann Arbor, Michigan, 48109-1048, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Angiosperms / enzymology*,  genetics
Base Sequence
Catalytic Domain / genetics
DNA Primers / genetics
Kinetics
Methyltransferases / chemistry*,  genetics,  metabolism*
Molecular Sequence Data
Mutagenesis, Site-Directed
Phylogeny
Sequence Homology, Amino Acid
Substrate Specificity
Chemical
Reg. No./Substance:
0/DNA Primers; EC 2.1.1.-/Methyltransferases; EC 2.1.1.-/eugenol O-methyltransferase; EC 2.1.1.68/caffeate O-methyltransferase

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


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