title: Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis.
abstract: Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).
authors.affiliation: Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
authors.forename: Brunella
authors.initials: B
authors.lastname: Felicetti
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D008401:Gas Chromatography-Mass Spectrometry; D006838:Hydrocarbons / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry; D013696:Temperature; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D006838:Hydrocarbons; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; D001224:Aspartic Acid; D014443:Tyrosine; D007535:Isomerases; C060050:aristolochene synthase; D008274:Magnesium