Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

Abstract

A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.

Fig. 1. Proposed biosynthesis of terpentecin.

a Structures of labdane (red), halimane (green), and clerodane (blue) skeletons. b The tpn BGC consists of two TSs (blue), two P450s (red), a ferredoxin (brown) and a GGPP synthase (green); mevalonate pathway genes responsible for production of the terpene precursors are found downstream. c Tpn2 first cyclizes GGPP into terpentedienyl diphosphate (2), a clerodane diterpene. Tpn3 eliminates the diphosphate of 2 to produce terpentetriene (3). The oxidation of 3 to terpentecin is still unknown. Carbon numbering for both 1 and 2 are shown. d HPLC analysis of the Tpn2 in vitro reaction confirms its type II TS activity. Negative control is reaction with boiled Tpn2. Products are shown as the dephosphorylated analogs.

Fig. 2. Structure of Tpn2.

a Overall βγ didomain structure of Tpn2 (PDB ID 7XKX, blue) aligned with PtmT2 (PDB ID 5BP8, yellow) and Rv3377c (PDB ID 6VPT, pink). The active site pocket of Tpn2 is circled in green. b Hydrophobic residues found in the active site pocket of Tpn2. The σA-weighted difference (mF_o − DF_c) omit map for Tpn2 with a 3σ contour is shown in green mesh. c Proposed binding mode of GGPP in the active site of Tpn2. C14 of GGPP lies 3.2 Å away from the carboxylate side chain of D296. G485, and the corresponding D502 in PtmT2, lie 3.6 Å from C19 of GGPP. Comparisons of the active site pockets of Tpn2 (d), PtmT2 (e), and Rv3377c (f).

Fig. 3. G485 is a single residue switch controlling labdane and clerodane formation in Tpn2.

a Cyclization mechanism of Tpn2 (blue) and Tpn2^G485D (red). b HPLC analysis of Tpn2-Tpn3 and Tpn2^G485D-Tpn3 fusion proteins in the E. coli MKI4 system.

Fig. 4. Bioinformatics of bacterial type II TSs.

The 964 identified type II TSs separate into four major families in an SSN at an e-value of 10⁻⁶⁰ (a) and six major families at an e-value of 10⁻⁸⁸ (b). Functionally characterized TSs producing labdane (yellow), clerodane (black), or halimane (blue) are outlined. Sequence logos highlighting the conservation of the DxDD and Asp/Gly-containing motifs are shown. The number of TSs in each family and taxonomic distributions are shown below the sequence logos.