Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Abstract

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.T erpenoid compounds form a large group of natural products synthesized by many species of plants, fungi, and bacteria. To date, more than 70,000 of these compounds have been identified, comprising ∼25% of all of the known natural products (Dictionary of Natural Products database, dnp.chemnetbase.com/intro/ index.jsp). Terpenoids and molecules containing terpenoid fragments are produced from two basic five-carbon building blocks: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1, 2). IPP and DMAPP are then converted into a series of terpenoid diphosphate esters of increasing chain lengths by chain length selective polyprenyl diphosphate synthases to give geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP, C 15 ), geranylgeranyl diphosphate (GGPP, C 20 ), and higher five-carbon homologs (3). These molecules are substrates for terpene synthases, which catalyze hydroxylation, elimination, cyclization, and rearrangement reactions to produce much of the structural diversity of terpenoid molecules found in nature (4, 5).The two main classes of terpene synthases (class I and class II) are distinguished from one another by the mechanisms they use to initiate carbocationic cyclization and rearrangement reactions and by their respective folds (6, 7). Class I terpene synthases use active site Mg 2+ ions to bind and activate the diphosphate moieties of their substrates as leaving groups to generate highly reactive allylic carbocations. In those terpene synthases that catalyze cyclization reactions, the carbocations alkylate distal double bonds in the hydrocarbon chain. The initial cycliza...

Structural and Chemical Biology of Terpenoid Cyclases

“…Importantly, successful attempts at predicting the product of a newly discovered sesquiterpene cyclase through computational approaches culminated in the recent discovery of cucumene synthase. 305 …”

Genome neighborhood network reveals insights into enediyne biosynthesis and facilitates prediction and prioritization for discovery

“…It is quickly evident which protein families are conserved for all enediynes, conserved based on associated peripheral moieties, unique to certain enediyne structures, and novel or unrelated to enediyne biosynthesis. As shown with protein similarity networks [4,8,56], outlining the nodes corresponding to functionally characterized proteins can help identify and prioritize protein families for future studies. For example, several genes (i.e., sgcJ and sgcM homologues) are fairly well conserved throughout both known and putative enediyne gene clusters, but their functions remain unknown.…”

Sesquiterpene Synthase–3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum

“…2) showed that HS appears to be similarly related to the bacterial STSs and to a small cluster of 1,6-and 1,10-cyclizing Ascomycota STSs (prAS, atAS, and FfSc6) (13) (12). Intriguingly, the bacterial STS clade includes several 1,11-cyclizing enzymes, including the recently discovered cucumene synthases (slt18 1880 and SCLAV p1407) that produce 1-and 4-cucumene (aka isohirsut-1-ene and isohirsut-4-ene, respectively) (33,34,53). The cucumane scaffold differs from the hirsutane scaffold only in the position of one methyl side group (2) as the result of a divergent cyclization path following the initial cyclization of FPP into the 1,11 trans-humulyl cation (20).…”