title: CdpC2PT, a reverse prenyltransferase from Neosartorya fischeri with a distinct substrate preference from known C2-prenyltransferases.
abstract: A putative prenyltransferase gene, NFIA_043650, was amplified from Neosartorya fischeri NRRL 181 and cloned into the expression vector pQE60. The deduced polypeptide consisting of 445 amino acids with a molecular mass of 51 kDa was overproduced in Escherichia coli and purified as His6-tagged protein to near homogeneity. The purified soluble protein was subsequently assayed with potential aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis of the reaction mixtures revealed acceptance of all tested tryptophan-containing cyclic dipeptides. Isolation and structural elucidation of enzyme products of five selected substrates indicated a reverse C2-prenylation on the indole nucleus, proving the enzyme to be a cyclic dipeptide C2-prenyltransferase (CdpC2PT). Differing significantly from two known brevianamide F reverse C2-prenyltransferases NotF and BrePT which use cyclo-l-Trp-l-Pro as their preferred substrate, CdpC2PT showed a clear substrate preference for (S)-benzodiazepinedinone and cyclo-l-Trp-l-Trp with KM values of 84.1 and 165.2 µM and turnover numbers at 0.63 and 0.30 s(-1), respectively. A possible role of CdpC2PT in the biosynthesis of fellutanines is discussed.
authors.affiliation: Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, 35032 Marburg, Germany.|Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Mundt
authors.affiliation: Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, 35032 Marburg, Germany.|Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.
authors.forename: Shu-Ming
authors.initials: SM
authors.lastname: Li
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004122:Dimethylallyltranstransferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055326:Neosartorya / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D004122:Dimethylallyltranstransferase