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.

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The class I sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS) catalyzes the transformation of linear farnesyl diphosphate (FPP) into the tricyclic hydrocarbon epi-isozizaene in the biosynthesis of albaflavenone antibiotics. The active site cavity of EIZS is largely framed by four aromatic residues – F95, F96, F198, and W203 – that form a product-shaped contour, serving as a template to chaperone conformations of the flexible substrate and multiple carbocation intermediates leading to epi-isozizaene. Remolding the active site contour by mutagenesis can redirect the cyclization cascade away from epi-isozizaene biosynthesis to generate alternative sesquiterpene products. Here, we present the biochemical and structural characterization of four EIZS mutants in which aromatic residues have been substituted with polar residues (F95S, F96H, F198S, and F198T) to generate alternative cyclization products. Most notably, F95S EIZS generates a mixture of monocyclic sesquiterpene precursors of bisabolane, a D2 diesel fuel substitute. X-ray crystal structures of the characterized mutants reveal subtle changes in the active site contour showing how each aromatic residue influences the chemistry of a different carbocation intermediate in the cyclization cascade. We advance that EIZS may serve as a robust platform for the development of designer cyclases for the generation of high-value sesquiterpene products ranging from pharmaceuticals to biofuels in synthetic biology approaches.

Copalyl diphosphate (CPP) synthase from Penicillium verruculosum (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α domain catalyzes the condensation of C5 dimethylallyl diphosphate with three successively added C5 isopentenyl diphosphates (IPPs) to form C20 geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ)6 hexamer. In the current study, we explore prenyltransferase structure–function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid products. Specifically, we report steady-state enzyme kinetics, product analysis, and crystal structures of various site-specific variants of the prenyltransferase α domain. Crystal structures of the H786A, F760A, S723Y, S723F, and S723T variants have been determined at resolutions of 2.80, 3.10, 3.15, 2.65, and 2.00 Å, respectively. The substitution of S723 with bulky aromatic amino acids in the S723Y and S723F variants constricts the active site, thereby directing the formation of the shorter C15 isoprenoid, farnesyl diphosphate. While the S723T substitution only subtly alters enzyme kinetics and does not compromise GGPP biosynthesis, the crystal structure of this variant reveals a nonproductive binding mode for IPP that likely accounts for substrate inhibition at high concentrations. Finally, mutagenesis of the catalytic general acid in the class II cyclase domain, D313A, significantly compromises prenyltransferase activity. This result suggests molecular communication between the prenyltransferase and cyclase domains despite their distant connection by a flexible polypeptide linker.

The magnificent chemodiversity of terpenoid natural products is largely rooted in two types of terpene synthases: prenyltransferases and cyclases. The unusual diterpene (C20) synthase, (+)‐copalyl diphosphate synthase from fungal Penicilliumspecies (PvCPS, PfCPS),is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain. The C‐terminal prenyltransferase α‐domain generates the C20 linear isoprenoid, geranylgeranyl diphosphate, which is then cyclized to (+)‐copalyl diphosphate at the interface of the N‐terminal βγ‐domains. We, first, establish that PvCPS exists as a hexamer – a unique quaternary structure for known αβγ terpene synthases.1 Hexamer assembly is corroborated by 1) crystal structures of the prenyltransferase α‐domain obtained from limited proteolysis of full‐length PvCPS, 2) the ab initio modeling of full‐length PvCPS derived from small‐angle X‐ray scattering data, and 3) preliminary 3D reconstructions of PfCPS by cryo‐EM. Interestingly, biophysical experiments with PvCPS suggest that oligomer formation is dynamic since the hexamer dissociates into lower‐order species at lower concentrations. However, enzyme concentration does not affect prenyltransferase activity in vitro. Assembly‐line catalysis provides an efficient carbon management system for generating high‐value terpenoid natural products. With the goal of generating different C5n isoprenoid products, we next explored the structure‐function relationship of the prenyltransferase domain within the assembly‐line platform of PvCPS. Steady‐state kinetics, product analysis, and crystal structures of various structure guided site‐directed variants afforded a functional C15 prenyltransferase chimera.2 This variant will be used in future engineering experiments to create bifunctional sesquiterpene synthases. References 1. Ronnebaum, T.A.; Gupta, K.; Christianson, D.W., J. Struct. Biol.2020, 210 (1), 107463. 2. Ronnebaum, T.A.; Eaton, S.A.; Brackhahn, E.A.E.; Christianson, D.W., Biochem. 2021, 60 (42), 3162‐3272.