Preparation of pyrrolo[2,3-b]indoles carrying a β-configured reverse C3-dimethylallyl moiety by using a recombinant prenyltransferase CdpC3PT

Abstract

Six beta-configured reversely C3-prenylated pyrrolo[2,3-b]indoles were successfully prepared by using a recombinant prenyltransferase from Neosartorya fischeri. For this purpose, the putative prenyltransferase gene NFIA_074280 (termed herewith cdpC3PT) was cloned into pQE60 and overexpressed in Escherichia coli. The overproduced His(6)-CdpC3PT was purified to near homogeneity and incubated with five cyclic tryptophan-containing dipeptides in the presence of dimethylallyl diphosphate (DMAPP). All of the substrates were accepted by CdpC3PT and converted to reversely C3-prenylated pyrrolo[2,3-b]indoles. Using cyclo-l-Trp-l-Trp as substrate, both mono- and diprenylated derivatives were obtained. The structures of the enzymatic products were confirmed by HR-ESI-MS, (1)H- and (13)C-NMR analyses as well as by long-range (1)H-(13)C connectivities in heteronuclear multiple-bond correlation (HMBC) spectra after preparative isolation. (1)H-(1)H spatial correlations in nuclear overhauser effect spectroscopy (NOESY) were used for determination of absolute configuration. The K(M) values were determined at about 1.5 mM for DMAPP and in the range from 0.22 to 5.5 mM for cyclic dipeptides. The turnover number k(cat) were found in the range of 0.023 to 0.098 s(-1) and specificity constants k(cat)/K(M) from 14.2 to 122.7 M(-1) s(-1). In contrast to the products of AnaPT bearing alpha-configured C3-dimethylallyl residues, the C3-prenyl moieties in the products of CdpC3PT have a beta-configuration. Discovery and characterisation of CdpC3PT expand the usage of the chemoenzymatic approach for stereospecific synthesis of C3-prenylated derivatives.

Prenyltransferases as key enzymes in primary and secondary metabolism

“…Tryptophan-containing cyclic dipeptide prenyltransferases catalyze regiospecific prenylations at different positions of the indole ring, especially at N-1, C-2, C-3, and C-7 (Grundmann and Li 2005;Wunsch et al 2015;Yin et al 2009Yin et al , 2010Yin et al , 2013aZou et al 2010). Some peptide-related substances like cyclo-acetoacetyl-L-tryptophan (cAATrp) or ardeemin FQ, a derivative of the cyclic tripeptide of anthranilic acid, alanine, and tryptophan, were also identified as substrates of this subgroup (Haynes et al 2013;Liu and Walsh 2009).…”

Biochemical Characterization of Indole Prenyltransferases

“…Indole prenyltransferases catalyze transfer reactions of prenyl moieties onto the indole nucleus and are involved in the biosynthesis of diverse natural products, especially mycotoxins (1,15). A large number of indole prenyltransferases, mainly belonging to the DMATS superfamily from fungi of Ascomycetes, have been identified in the last years and characterized biochemically (1,10,11). The members of the DMATS superfamily are soluble proteins, showed usually broad substrate specificity and accepted tryptophan, simple indole derivatives, tryptophan-containing cyclic dipeptides, or other indole-containing structures as aromatic substrates and DMAPP as prenyl donor (1,10,11).…”

“…A large number of indole prenyltransferases, mainly belonging to the DMATS superfamily from fungi of Ascomycetes, have been identified in the last years and characterized biochemically (1,10,11). The members of the DMATS superfamily are soluble proteins, showed usually broad substrate specificity and accepted tryptophan, simple indole derivatives, tryptophan-containing cyclic dipeptides, or other indole-containing structures as aromatic substrates and DMAPP as prenyl donor (1,10,11). A few examples of soluble indole prenyltransferases have also been identified in bacteria (13,45).…”

Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

“…However, no products were formed with DMAPP as the prenyl donor. We also used several cyclo-dipeptides and hydroxynaphthalenes as substrates since they were reported to be utilized by many fungal prenyltransferases (Grundmann and Li 2005;Yin et al 2007;Yin et al 2010;Yu et al 2011;Zou et al 2010). However, no products were formed.…”