Biology subjects

Gaynes, M. N.

Publications and source records attributed to Gaynes, M. N..

3 recordsLinked to original sources

bioRxiv2025

Structure and Mechanism of Avermitilol Synthase, a Sesquiterpene Cyclase that Generates a Highly Strained 6-6-3 Tricyclic Alcohol

Avermitilol synthase from Streptomyces avermitilis (SaAS) is a high-fidelity class I terpene cyclase that converts farnesyl diphosphate into a highly-strained, 6-6-3 tricyclic sesquiterpene alcohol. The mechanism of avermitilol formation proceeds through a 10-3 bicyclic intermediate, bicyclogermacrene, which undergoes proton-initiated anti-Markovnikov opening of two separate C=C bonds in a transannulation mechanism that forms the 6-6-3 tricyclic skeleton, with quenching by water to yield avermitilol. Small amounts of a side product, viridifloral, result from Markovnikov opening of one of the reactive C=C bonds. Here, we present enzymological studies of SaAS to establish the substrate scope and metal ion dependence for catalysis, and we present crystal structures of SaAS complexed with a variety of ligands that partially mimic carbocation intermediates in catalysis. Interestingly, these structures show that two water molecules remain trapped in a polar crevice in the active site regardless of the ligand bound. Structure-activity relationships for site-specific mutants yield key insight on the catalytic importance of these trapped water molecules. Specifically, T215 normally hydrogen bonds with water molecule W1, but the T215V substitution breaks this hydrogen bond and causes W1 to shift by 1.3 [A] to form a hydrogen bond with W300. Avermitilol generation is completely blocked in this mutant, but the generation of viridifloral and another side product is enhanced. Thus, the T215V substitution causes water molecule W1 to align for reaction with the tertiary and not the secondary carbon in the reactive C=C bond of bicyclogermacrene. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/675706v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@172e90corg.highwire.dtl.DTLVardef@1223258org.highwire.dtl.DTLVardef@1751d6org.highwire.dtl.DTLVardef@fdf739_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Cryo-EM Structure of the Cyclase Domain and Evaluation of Substrate Channeling in a Bifunctional Class II Terpene Synthase

Copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. The various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure-function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 [A]-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core, but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase-cyclase association facilitates substrate channeling due to active site proximity.

biochemistry↗

bioRxiv2025

Crystal Structure and Catalytic Mechanism of Drimenol Synthase, a Bifunctional Terpene Cyclase-Phosphatase

Drimenol synthase from Aquimarina spongiae (AsDMS) is a highly unusual bifunctional sesquiterpene synthase that integrates two distinct, sequential isoprenoid processing activities within a single polypeptide chain. AsDMS catalyzes the class II cyclization of farnesyl diphosphate (FPP) to form drimenyl diphosphate, which then undergoes enzyme-catalyzed hydrolysis to yield drimenol, a bioactive sesquiterpene alcohol with antifungal and anticancer properties. Here, we report the X-ray crystal structures of AsDMS and its complex with a sesquiterpene thiol, which are the first of a terpene cyclase-phosphatase. The AsDMS structure exhibits a two-domain architecture consisting of a terpene cyclase {beta} domain and a haloacid dehalogenase (HAD)-like phosphatase domain, with two distinct active sites located on opposite sides of the protein. Mechanistic studies show that dephosphorylation of the drimenyl diphosphate intermediate proceeds through stepwise hydrolysis, such that two equivalents of inorganic phosphate rather than inorganic pyrophosphate are co-products of the reaction sequence. When the AsDMS reaction is performed in H218O, 18O is not incorporated into drimenol, indicating that the hydroxyl oxygen of drimenol originates from the prenyl oxygen of FPP rather than bulk water. These results correct a mechanistic proposal previously advanced by another group. Surprisingly, AsDMS exhibits unprecedented substrate promiscuity, catalyzing the conversion of substrate mimic farnesyl-S-thiolodiphosphate into cyclic and linear sesquiterpene products. Structural and mechanistic insights gained from AsDMS expand the functional diversity of terpene biosynthetic enzymes and provide a foundation for engineering "designer cyclases" capable of generating new terpenoid products.

biochemistry↗