Structural Basis of Substrate Promiscuity and Catalysis by the Reverse Prenyltransferase N-Dimethylallyl-l-tryptophan Synthase from Fusarium fujikuroi

Abstract

The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-L-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C−C, C−N, and C−O bond-forming trajectories. DMATS1 from Fusarium f ujikuroi catalyzes the reverse N-prenylation of L-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of L-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or L-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C−N and C−O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with L-Trp or L-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of L-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure−function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.

Structure‐Guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6‐Indole Prenyltransferases

“…These positions are occupied by aromatic amino acids in different wild‐type enzymes (Table S5). The conserved Tyr in position 2 seems to play a role in binding the acceptor, and mutation to Ala led to a four‐fold reduction in DMATS1 activity [28] . Only in IPTs that catalyze C6 prenylation, there is a strict need for one of those two residues to be His to act as a catalytic base.…”

“…The Tyr364 hydroxyl group is too far to propose a role in the catalysis of C6 (Figure 2B). Structural alignment of PriB (C6) with selected non‐C6 IPTs [DMATS1 (PDB ID 8DB0, N‐1), [28] AmbP3 (PDB ID 5Y7C, C2), FtmPT1 (PDB ID 3O2K, C3 rearranges to C‐2), [29,30] CdpNPT (PDB ID 4E0U, C3 rearranges to N1), [31,32] AnaPT (PDB ID 4LD7, C3), [33] FgaPT2 (PDB ID 3I4X, C4), [27] 5DMATSsc (PDB ID 6ZRZ, C5), [26] MpnD (PDB ID 4YLA, C7) [34] ] reveal that His312 in PriB replaces a conserved Tyr residue that is present in non C6 IPTs (Figure 2C, Figures S3–S4). Recently, a His residue was determined as the key catalytic residue for 6DMATSmo [26] .…”

“…Our work shows that structure‐guided mutagenesis of His in this region alters the regiospecificity of C6 enzymes. The regiospecificity of IPTs appears to be determined by various factors; i) presence of a catalytic residue at an appropriate distance from the targeted indole position as in the case with Glu and N1 [28] and C2, [29] Lys and C4, [27] Gln and C5 [26] and His in C6 as characterized in this study and others [26] . The function of this residue is to abstract proton, restore aromaticity and reduce activation energy; ii) distance of the catalytic residue from the position to be prenylated; iii) relative orientation and proximity between C1′ or C3′ of the donor and the targeted carbon position at the acceptor within the IPT binding pocket; iv) potential rearrangement as in the case of CdpNPT and FtmPT1; [30,32] v) nature and size of the substrates that can cause steric hindrance and lead to subsequent shift in the active site; [19,37] vi) potential participation of active site waters as proposed with Gln and 5DMATSsc [26] .…”

“…The release of the pyrophosphate moiety from 2 generates an allylic carbocation 2 a. A strictly conserved glutamate residue [24,27,28] in IPTs (Glu94 in PriB) positioned at close proximity to N1 forms a hydrogen bond with the indole NÀ H. This hydrogen bond increases the indole ring reactivity towards electrophilic substitution by 2 a at the 1 C6 position. [27] The σ-complex intermediate 1 a is stabilized via charge complementation.…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…Three‐dimensional structural analysis and engineering of IPTs have revealed insights into the promiscuity of several members of this class. The size of the active site as well as the presence and absence of specific residues control substrate specificity [198,204,206] . In addition, rational design of IPTs has led to the generation of variants with increased promiscuity, [207] change of regiospecificity, [203,205,208] or altered donor preference [209] .…”