Structural Basis of Tryptophan Reverse N-Prenylation Catalyzed by CymD

Abstract

Indole prenyltransferases catalyze the prenylation of Ltryptophan (L-Trp) and other indoles to produce a diverse set of natural products in bacteria, fungi, and plants, many of which possess useful biological properties. Among this family of enzymes, CymD from Salinispora arenicola catalyzes the reverse N1 prenylation of L-Trp, an unusual reaction given the poor nucleophilicity of the indole nitrogen. CymD utilizes dimethylallyl diphosphate (DMAPP) as the prenyl donor, catalyzing the dissociation of the diphosphate leaving group followed by nucleophilic attack of the indole nitrogen at the tertiary carbon of the dimethylallyl cation. To better understand the structural basis of selective indole N-alkylation reactions in biology, we have determined the X-ray crystal structures of CymD, the CymD−L-Trp complex, and the CymD−L-Trp−DMSPP complex (DMSPP is dimethylallyl S-thiolodiphosphate, an unreactive analogue of DMAPP). The orientation of L-Trp with respect to DMSPP reveals how the active site contour of CymD serves as a template to direct the reverse prenylation of the indole nitrogen. Comparison to PriB, a C6 bacterial indole prenyltransferase, offers further insight regarding the structural basis of regioselective indole prenylation. Isothermal titration calorimetry measurements indicate a synergistic relationship between L-Trp and DMSPP binding. Finally, activity assays demonstrate the selectivity of CymD for L-Trp and indole as prenyl acceptors. Collectively, these data establish a foundation for understanding and engineering the regioselectivity of indole prenylation by members of the prenyltransferase protein family.

Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1Ff

“…A recently published crystal structure of substrate bound CymD strongly suggests a direct nucleophilic attack also in that case . Despite the similar reaction mechanisms, the active site architectures of CymD and DMATS1 Ff disclose strong differences.…”

“…Despite the similar reaction mechanisms, the active site architectures of CymD and DMATS1 Ff disclose strong differences. While for DMATS1 Ff binding of the substrates results in a similar aromatic cavity as in FgaPT2 (Figure S15), DMAPP and tryptophan are found with inverted relative orientation in the CymD crystal structure . Further, CymD does not seem to coordinate significantly to the polar residues of the amino acid side chain.…”

Recent Advances in the Biosynthesis of Carbazoles Produced by Actinomycetes

“…PTs can be categorized depending on their primary sequence similarity and on their substrates as aromatic acceptors for prenylation (i.e., bacterial phenol/phenazine PTs, fungal or bacterial indole PTs, and membrane-bound aromatic PTs) ( Figure 6). The subgroup of soluble aromatic or indole PTs contains an (ααββ) 4 -(αββα) structural motif (named PT-barrel fold); for example, NphB [49], FgaPT2 [50], and CymD [51], designated as ABBA-type PTs. The PT-barrel fold was discovered during structural elucidation of NphB, which is a key enzyme for naphtherpin biosynthesis [49].…”

Genome‐Mining‐Based Discovery of the Cyclic Peptide Tolypamide and TolF, a Ser/Thr Forward O‐Prenyltransferase

“…[8b] A recent structure of a non-cyanobactin enzyme, the Trp N-prenyltransferase, CymD showed that the C-3 carbon of the dimethylallyl group is 2.5 closer to the substrate than the C-1 carbon, leading to facile reverse prenylation. [24] We expect similar structural nuances give rise to different regiospecific-ities of TolF and TruF1, although their relatively distant sequence similarity makes direct comparison challenging ( Figure S16).…”

“…In the PagF (forward Tyr prenylating) structure, phenolic oxygen of the substrate is 1 Å closer to the C‐1 carbon atom of dimethylallyl group than the C‐3 carbon, resulting in favourable O ‐prenylation of Tyr [8b] . A recent structure of a non‐cyanobactin enzyme, the Trp N ‐prenyltransferase, CymD showed that the C‐3 carbon of the dimethylallyl group is 2.5 Å closer to the substrate than the C‐1 carbon, leading to facile reverse prenylation [24] . We expect similar structural nuances give rise to different regiospecificities of TolF and TruF1, although their relatively distant sequence similarity makes direct comparison challenging (Figure S16).…”