title: The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases.
abstract: Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.); birgit.piechulla@uni-rostock.de.|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and birgit.piechulla@uni-rostock.de.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.) birgit.piechulla@uni-rostock.de.
authors.forename: Birgit
authors.initials: B
authors.lastname: Piechulla
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Richard
authors.initials: R
authors.lastname: Bartelt
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Anne
authors.initials: A
authors.lastname: Brosemann
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Uta
authors.identifier: 0000-0002-2744-6796
authors.initials: U
authors.lastname: Effmert
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Harro
authors.initials: H
authors.lastname: Bouwmeester
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Frank
authors.initials: F
authors.lastname: Hippauf
authors.affiliation: Institute of Biological Sciences, Biochemistry, University of Rostock, 18059 Rostock, Germany (B.P., A.B., U.E., F.H.).|Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), Germany (R.B., W.B.); and.|Plant Sciences, University of Wageningen, 6708PB Wageningen, The Netherlands (H.B.).
authors.forename: Wolfgang
authors.identifier: 0000-0002-0825-1491
authors.initials: W
authors.lastname: Brandt
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003198:Computer Simulation; D003500:Cyclization; D000081005:Cyclohexane Monoterpenes; D003511:Cyclohexanols / Q000737:chemistry / Q000378:metabolism*; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism*; D000077591:Eucalyptol; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D040681:Structural Homology, Protein; D014026:Nicotiana / Q000201:enzymology*; D055549:Volatile Organic Compounds / Q000032:analysis
chemical_list: D000081005:Cyclohexane Monoterpenes; D003511:Cyclohexanols; D053138:Cyclohexenes; D039821:Monoterpenes; D050505:Mutant Proteins; D055549:Volatile Organic Compounds; C016775:alpha-terpineol; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D000077591:Eucalyptol