Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site.
Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Delta24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.
Nes
W D
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Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA. wdavid.nes@ttu.edu
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004798:Enzymes / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000378:metabolism*; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D004798:Enzymes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D008780:Methyltransferases; C474634:SMT1 protein, Arabidopsis