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Review
. 2019 Oct 2:15:2355-2368.
doi: 10.3762/bjoc.15.228. eCollection 2019.

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

Affiliations
Review

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

Ronja Driller et al. Beilstein J Org Chem. .

Abstract

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

Keywords: CotB2; biotechnology; crystal structure; cyclooctatin; diterpene; reaction mechanism; terpene synthase.

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Figures

Figure 1
Figure 1
CotB1 synthesizes geranylgeranyl diphosphate (GGDP) 3 from the substrates dimethylallyl diphosphate (DMAPP) 1 and isopentenyl diphosphate (IPP) 2. The acyclic substrate GGDP (3) is stereospecifically cyclized by CotB2 to cyclooctat-9-en-7-ol (4), with a fusicoccane 5–8–5 fused ring system. Two cytochrome P450 enzymes, CotB3 and CotB4, subsequently functionalize cyclooctat-9-en-7-ol (4) to the bioactive compound cyclooctatin (5).
Figure 2
Figure 2
The bacterial diterpene synthase CotB2wt·Mg2+3·F-Dola in the closed, active conformation (PDB-ID 6GGI; [37]). (A) The two monomers of CotB2 are shown in cartoon representation with the α-helices drawn as cylinders and colored in light brown. One monomer of CotB2 is shown in gray surface representation. The location of the aspartate-rich motif is indicated in red and the NSE-motif is marked in yellow. The WXXXXXRY motif is indicated in light-green. The last 12 C-terminal residues of the lid are drawn in violet. The three Mg2+ ions are represented as green spheres and the bound intermediate F-Dola is shown in magenta. The cleaved diphosphate is shown in orange. (B) View of panel A rotated by 45°. For clarity, one monomer has been removed. The view is into the active site of CotB2.
Figure 3
Figure 3
Conformational changes of CotB2 upon ligand binding. Superposition of CotB2’s open (teal), pre-catalytic (black, CotB2wt·Mg2+B·GGSDP), and fully closed (light-brown, CotB2wt·Mg2+3·F-Dola) conformation. The overall fold of CotB2wt·Mg2+B·GGSDP (PDB-ID 5GUE; [36]) is more similar to CotB2wt (PDB-ID 4OMG; [38]) than to CotB2wt·Mg2+3·F-Dola (PDB-ID 6GGI; [37]). The salt bridge between D111 and R294, in stick representation, is shown by red, dashed lines. Mg2+ ions are colored in green. Black arrows indicate movement of secondary structure elements from the open to the closed conformation of CotB2. The different C-termini are labeled in all three structures. The C-terminus in the structure of CotB2wt·Mg2+3·F-Dola is colored in purple.
Figure 4
Figure 4
View into the active site of CotB2wt·Mg2+3·F-Dola [37] superimposed with CotB2wt·Mg2+B·GGSDP [36]. (A) The bound F-Dola reaction intermediate is shown in magenta with the fluorine atom colored in light blue, and Mg2+ions are shown in green. Residues of the DDXD motif are shown in red and residues of the NSE motif in yellow. The conserved residues of the WXXXXXRY motif are drawn in light green and the R227 that interacts with diphosphate moiety in light brown. The cleaved diphosphate is shown in orange. Gray lines represent the coordination sphere of the Mg2+ ions. Identical amino acid residues located in the structure of CotB2wt·Mg2+B·GGSDP are shown in gray. The GGSDP molecule is shown in black with the sulfur atom colored in yellow. The position of Mg2+B is identical in both structures. For clarity water molecules have been omitted. (B) View in panel A rotated by 30°. In addition to the motifs shown in panel A, the pyrophosphate sensor motif is depicted in pink.
Figure 5
Figure 5
View into the active site of CotB2wt·Mg2+3·F-Dola [37]. Identical view as in Figure 4. (A) The bound F-Dola reaction intermediate is shown in magenta with the fluorine atom colored in light blue, and Mg2+-ions are shown in green. Residues of the DDXD motif are shown in red and residues of the NSE motif in yellow. The conserved residues of the WXXXXXRY motif are drawn in light green and the R227 that interacts with diphosphate moiety in light brown. The cleaved diphosphate is shown in orange. Hydrogen bonds and salt bridges are indicated by dashed lines in the same color-coding as the involved motifs. Gray lines represent the coordination sphere of the Mg2+-ions. For clarity water molecules have been omitted. (B) View in panel A rotated by 30°. In addition to the motifs shown in panel A, the pyrophosphate sensor motif is depicted in pink.
Figure 6
Figure 6
The WXXXXXRY motif in protein sequences of diterpene TPS from different bacteria. Highlighted is the WXXXXXRY motif in green. Underlined sequences refer to crystal structures of the respective diterpene TPS in the closed conformation, with a structured C-terminus. Cyclooctat-9-en-7-ol synthase (WP_093468823), S. melanosporofaciens; labdane-related diterpene synthase (WP_019525557), Streptomyces K155; iso-elsabellatriene synthase (WP_003963279), Streptomyces clavuligerus; terpene synthase (WP_046708564.1), Streptomyces europaeiscabiei; terpene synthase (WP_012394883), Myobacterium marinum; diterpene synthase (BAP82229), Streptomyces sp. ND90; tsukubadiene synthase (EIF90392), Streptomyces tsukubaensis NRRL 18488; terpene synthase (ZP_00085244), Pseudomonas fluorescens PfO-1; spiroalbatene synthase (WP_030426588.1), Allokutzneria albata; spatadien synthase (WP_095757924.1) Streptomyces xinghaiensis.
Scheme 1
Scheme 1
Overview of the altered product portfolio as a result of introduced point mutations in the active site of CotB2. For an overview of the point mutations see Table 2.
Scheme 2
Scheme 2
Catalytic mechanism of CotB2, derived from isotope labeling experiments [–35], density functional theory calculations [33] as well as QM/MM simulations [37].
Figure 7
Figure 7
(A) The inner surface of the active site is shown in gray. The bound F-Dola reaction intermediate is shown in magenta with the fluorine atom colored in light blue. Aromatic residues are shown in black stick representation (B) View of panel A rotated by 90°. (C) View from the top into the active site of CotB2. View of panel A rotated by 90°.
Scheme 3
Scheme 3
Variants of CotB2 open the route to a novel product portfolio with altered cyclic carbon skeletons, which can be converted into bioactive compounds by chemo-enzymatic methodologies. Modification descriptions are composed according to the atom numbering. (A) Lead structure for the dolabellane derivatives is 3,7,18-dolabellatriene (12) and (B) cembrene A (7) for the cembranoid family.

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