First trans-eunicellane terpene synthase in bacteria

Abstract

Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

Keywords: Bacterial terpenoids; diterpenoid; enzymes; eunicellane; genome mining; isotope labeling; mechanism; quantum chemical calculations; terpene synthase.

Figure 1.. Eunicellane diterpenoid natural products and biosynthesis

(A) Selected members of eunicellane diterpenoids. The 6/10-bicyclic eunicellane skeleton possesses either cis or trans ring configuration and is found in coral, bacteria, and plants. The trans ring fusion is rare in known natural products. (B) Recent studies support mechanistic differences between two cis-eunicellane di-TSs, Bnd4 from Streptomyces sp. (CL12–4) and EcTPS1 from Erythropodium caribaeorum. Prior to this study, no TSs that form the trans-eunicellane skeleton were known.

Figure 2.. Sequence analysis highlights functional diversity of bacterial terpene synthases

Sequence similarity network of TSs (IPR034686) from bacteria at an e-value threshold of 10⁻⁷⁰. Functionally characterized TSs are colored (magenta, C15; blue, C20; blue with yellow highlight, AlbS) and their major products are shown. For clarity, not all characterized TSs are shown and not all products are shown for all clusters. In this enzyme family, there are 101 clusters of uncharacterized subfamilies and 151 uncharacterized singletons (not shown, see also Fig. S1). (inset) The AlbS subfamily of TSs and putative BGCs of AlbS and its homologues (see also Fig. S2). Genes are colored according to proposed function; green, GGPP synthase; cyan, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; red, cytochrome P450; brown, conserved protein of unknown function; gray, unrelated.

Figure 3.. Enzymatic activity of AlbS and structurally determined sesquiterpene and diterpene products

(A) GC-MS analysis of AlbS and AlbS^Y214F reactions with FPP. The y-axis is the relative abundance of total ions. (B) Enzymatic products identified in this study. See also Figs. S4–S22 and S62–S69. Only key 2D NMR correlations are shown for 1. See also Table S4. (C) HPLC-UV analysis of AlbS and mutant reactions with GGPP; 1 was also produced in vivo in albS-expressing E. coli. Absorbance was detected at 210 nm. See also Fig. S61. (D) Structural elucidation of 1, 8, 9, and 10 using NMR spectroscopy, chemical degradation, and Mosher’s analysis. See also Figs. S23–S60.

Figure 4.. Structural and mutational analysis of the AlbS active site

(A) Structural models of AlbS and Bnd4 displaying key active site residues (sticks) with a docked model of GGPP (gray ball and stick). The three Mg²⁺ ions (green spheres) from selinadiene synthase (PDB ID 4OKZ) were overlaid to show their approximate positions. (B) Relative cyclization activities of AlbS and mutants forming 1. Values are the mean of three independent experiments, which are shown as overlaid orange circles, with error bars representing standard deviations. The absence of a bar denotes no activity detected; NS denotes not soluble. Mutants marked with asterisks produced other products (see also Figs. 3 and S61).

Figure 5.. Mechanistic proposal and deuterium labeling support for the cyclization of GGPP into 1

(A) ¹H-¹³C HSQC spectra of 1, 1,11-²H₂-1, 11-²H-1, and 1-²H-2, respectively. See also Figs. S9, S72, and S74–S76. (B) Proposed mechanism for the formation of the trans-6,10-bicyclic eunicellane skeleton by AlbS (blue pathway). AlbS mutants yielding shunt products 13 and 14 are also shown (black pathways). The red R group depicts the location of the 1R-²H-labeling experiment. (C) Structure of 1-²H-2 from the 1R-²H-labeling experiment with Bnd4 supporting a different hydride transfer in the AlbS and Bnd4 mechanisms. (D) Relative free energies of intermediates and transition state structure in kcal mol⁻¹, calculated at mPW1PW91/6–31+G(d,p)//B3LYP/6–31+G(d,p) level of theory.^– Bond distances (in Å) for key steps are listed beside the bond. The conformations depicted here are qualitative; see computed structures for actual conformations.