title: Characterization of cyclo-acetoacetyl-L-tryptophan dimethylallyltransferase in cyclopiazonic acid biosynthesis: substrate promiscuity and site directed mutagenesis studies.
abstract: The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca(2+)-ATPase with a unique pentacyclic indole tetramic acid scaffold, is assembled by a three enzyme pathway CpaS, CpaD, and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form beta-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines, and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with 11 homologous fungal Trp-DMATs revealed five regions of conservation, suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Liu
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001231:Aspergillus flavus / Q000201:enzymology / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D002413:Cations, Divalent / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics; D054659:Diketopiperazines / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D055503:Protein Multimerization / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011760:Pyrrolidinones / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013867:Thiohydantoins / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D054659:Diketopiperazines; D004151:Dipeptides; D007211:Indoles; D011106:Polyisoprenyl Phosphates; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D013867:Thiohydantoins; C009435:tetramic acid; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase; C000543:cyclopiazonic acid