title: Substrate flexibility and reaction specificity of tropinone reductase-like short-chain dehydrogenases.
abstract: Annotations of protein or gene sequences from large scale sequencing projects are based on protein size, characteristic binding motifs, and conserved catalytic amino acids, but biochemical functions are often uncertain. In the large family of short-chain dehydrogenases/reductases (SDRs), functional predictions often fail. Putative tropinone reductases, named tropinone reductase-like (TRL), are SDRs annotated in many genomes of organisms that do not contain tropane alkaloids. SDRs in vitro often accept several substrates complicating functional assignments. Cochlearia officinalis, a Brassicaceae, contains tropane alkaloids, in contrast to the closely related Arabidopsis thaliana. TRLs from Arabidopsis and the tropinone reductase isolated from Cochlearia (CoTR) were investigated for their catalytic capacity. In contrast to CoTR, none of the Arabidopsis TRLs reduced tropinone in vitro. NAD(H) and NADP(H) preferences were relaxed in two TRLs, and protein homology models revealed flexibility of amino acid residues in the active site allowing binding of both cofactors. TRLs reduced various carbonyl compounds, among them terpene ketones. The reduction was stereospecific for most of TRLs investigated, and the corresponding terpene alcohol oxidation was stereoselective. Carbonyl compounds that were identified to serve as substrates were applied for modeling pharmacophores of each TRL. A database of commercially available compounds was screened using the pharmacophores. Compounds identified as potential substrates were confirmed by turnover in vitro. Thus pharmacophores may contribute to better predictability of biochemical functions of SDR enzymes.
authors.affiliation: Institute of Pharmacy, Faculty of Science I, Martin Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Reinhardt
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Fischer
authors.affiliation: Institute of Pharmacy, Faculty of Science I, Martin Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Coppi
authors.affiliation: Institute of Pharmacy, Faculty of Science I, Martin Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Blum
authors.affiliation: Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Brandt
authors.affiliation: Institute of Pharmacy, Faculty of Science I, Martin Luther University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle, Germany. Electronic address: birgit.draeger@pharmazie.uni-halle.de.
authors.forename: Birgit
authors.initials: B
authors.lastname: Dräger
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D019607:Brassicaceae / Q000201:enzymology; D020134:Catalytic Domain; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014326:Tropanes / Q000737:chemistry
chemical_list: D014326:Tropanes; D009243:NAD; C016808:3-tropinone; D000429:Alcohol Oxidoreductases; C083753:tropinone reductase
keywords: NAD(P)(H) specificity; Protein structure homology modeling; SDR; Tropinone reductase-like enzyme; Virtual screening