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

Chow J, Tian B, Ramamoorthy G, Hillerich BS, Seidel RD, Almo SC, Jacobson MP, Poulter CD, (2015) Proc Natl Acad Sci, 112, 5661-5666. PMCID: PMC4426440

In recent work from the IS Bridging Project and Computation Core, an innovative computational work-flow was capable of predicting the identities of the substrate, carbocation intermediate, and product carbon skeleton for an uncharacterized Terpene Synthase. The top ranked compound proved to be correct, therein demonstrating the superb accuracy of this predictive process despite the promiscuous nature of Terpene Synthase chemistry as well as the large enumeration of potential products. The IS Bridging Project was essential in characterizing both the enzyme activity, as well as the structure of the sesquiterpene product, (5S,7S,10R,11S)-cucumene.

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.

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Figure Figure 1: Sequence similarity network of terpen synthase-like 2 subgroup (A) with an e-value of 10^-50 and (B) zoom in at cluster 3 with an e-value of 10^-75.

Figure Figure 2: Computational workflow for the prediction of terpene synthase products.

Figure Figure 3: Top 10 predicted skeletons for 3LG5, Q55012, and B5GLM7. The number in the parentheses indicates the number of enumerated carbocations for each skeleton.

Figure Figure 4: Two- and three-dimensional structure of (5S,7S,10R,11S)-cucumene.

Reprinted with permission from Chow et al. PNAS. Copyright 2015 National Academy of Sciences.