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Review
. 2012 Jan 27;51(5):1124-37.
doi: 10.1002/anie.201103110. Epub 2011 Nov 21.

Terpene Biosynthesis: Modularity Rules

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Free PMC article
Review

Terpene Biosynthesis: Modularity Rules

Eric Oldfield et al. Angew Chem Int Ed Engl. .
Free PMC article

Abstract

Terpenes are the largest class of small-molecule natural products on earth, and the most abundant by mass. Here, we summarize recent developments in elucidating the structure and function of the proteins involved in their biosynthesis. There are six main building blocks or modules (α, β, γ, δ, ε, and ζ) that make up the structures of these enzymes: the αα and αδ head-to-tail trans-prenyl transferases that produce trans-isoprenoid diphosphates from C(5) precursors; the ε head-to-head prenyl transferases that convert these diphosphates into the tri- and tetraterpene precursors of sterols, hopanoids, and carotenoids; the βγ di- and triterpene synthases; the ζ head-to-tail cis-prenyl transferases that produce the cis-isoprenoid diphosphates involved in bacterial cell wall biosynthesis; and finally the α, αβ, and αβγ terpene synthases that produce plant terpenes, with many of these modular enzymes having originated from ancestral α and β domain proteins. We also review progress in determining the structure and function of the two 4Fe-4S reductases involved in formation of the C(5) diphosphates in many bacteria, where again, highly modular structures are found.

Figures

Figure 1
Figure 1
a) Structure of human FPPS showing conserved DDXXD motifs (red), Mg2+ (blue) and IPP (bottom) and S-thiolo DMAPP (top) ligands. b) expansion of the active site region in a, cationic residues in red and cyan. c) Hetero-tetramer structure of M. piperata GPPS showing catalytic α (yellow) and regulatory (δ) subunits. d) Superposition of the α,δ domains in GPPS. e) Zoledronate and IPP bound to the active site of human FPPS: color code as in a. f) Zoledronate and 19 (NOV-980) bound to the allylic (ZOL) and allosteric (NOV-980) sites in human FPPS.
Figure 2
Figure 2
Structures of GGPPS. a) Superposition of human FPPS (green) and human GGPPS (cyan) with the Asp-rich domain (red) and Mg2+ ions (blue) high-lighted as spheres. b) Zoledronate (ZOL) and IPP bound to active site in Plasmodium vivax GGPPS. c) Compound 21 (BPH-715, pink), IPP, Mg2+ bound to S. cerevisae GGPPS superimposed on GGPP (cyan) bound to the product site. Human GGPPS has a very similar local structure and is potently inhibited by 21, but not by zoledronate.
Figure 3
Figure 3
Structures of CrtM and SQS. a) S. aureus CrtM (green) with FSPP, Mg2+ superimposed on human SQS (orange): essential Asp residues and Mg2+ colored as in Fig. 1. b) Active site region in CrtM + FSPP (green, yellow), Mg2+. c) PSPP, Mg2+ (all in cyan) bound to CrtM, superimposed on FSPP/Mg2+ structure (in green/yellow/blue). d) Dehydrosqualene product (pink) bound to CrtM, superimposed on PSPP structure (cyan). S1 = allylic binding site; S2 = homoallylic binding site.
Figure 4
Figure 4
Genesis and evolution of terpene cyclases. a) Genes for ancestral βγ domain proteins (like SHC) fuse with genes for ancestral α-domain species like FPPS b) to generate αβγ three-helical domain diterpene cyclase, c) Orange shading indicates close proximity (~2.5 Å) of SHC C-terminus and FPPS N-terminus (from an α/αβ/βγ FPPS/EAS/SHC alignment). d) Structure of an actual diterpene cyclase, taxadiene synthase[51]. e) Loss of the γ domain yields an αβ protein, e.g. the sesquiterpene cyclase isoprene synthase. f) Further loss of the β domain yields other cyclases, e.g., pentalenene synthase, an α domain cyclase. Ancestral α and βγ-domain species presumably produced the FPP, GGPP and squalene used to produce lipids in Archaea; the αβγ~ derived families are much later arrivals. Note the N-terminal helix (magenta) portion is conserved in αβ, βγ, and αβγ proteins and is known to be required for activity.
Figure 5
Figure 5
Dimeric quaternary structure of three α2β2 terpene synthases. a) Limonene synthase and product limonene. b) Bornyl diphosphate synthase and product bornyl diphosphate. c) Isoprene synthase and product isoprene. The catalytic, α or C-terminal domains are in blue, the β or N-terminal domains are in green. The catalytic DDXXD domains are in red. The buried surface areas that comprise the dimerization interface are large, 1148 +/−88 A2. The Cα rmsd between three structures is 1.4-Å. This figure is adapted from Figure 8 in ref. and was constructed from the Protein Data Bank entries: 2ONG, 1N1B, and 3N0F.
Figure 6
Figure 6
Structures and dynamics of the ζ prenyl transferase UPPS. a) Overall structure of a bisphosphonate-bound E. coli UPPS monomer. b) substrates (FPP and IPP) bound to UPPS active site. c) Structural alignment of the predicted structure of Rv3378c with UPPS. d) Molecular dynamics simulation of UPPS. Black, data taken every 10 ps; grey, every 100 ps. e) frequency of occurence of pocket versus pocket volume. The apo structure has a small pocket volume; the largest volume is close to that occupied by a large inhibitor.
Figure 7
Figure 7
Modular structures of the 4Fe-4S cluster-containing proteins IspH and IspG. a) E. coli IspH showing the three helix/sheet domains surrounding the 4Fe-4S cluster in the “closed” form (which buries the Fe/S cluster). b) E. coli IspG showing an “open” structure. The 4Fe-4S cluster from one chain is thought to interact with the TIM barrel in the second chain to form the active site (black box). c) Superposition of the TIM barrel in E. coli IspG (orange) with B. anthracis dihydropteroate synthase (cyan). d) Superposition of the 4Fe-4S cluster domain in IspG with that in spinach nitrite reductase. The Cα rmsds in c,d are 2.4, 2.1 Å, respectively.
Figure 8
Figure 8
Schematic illustration summarizing the modular nature of many terpene/isoprenoid biosynthesis enzymes. FPPS and GPPS are αα dimers (human GGPPS a trimer of dimers); GPPS forms a heterotetramer α2δ2; plant diterpene cyclases (like TXS) are αβγ; many other plant terpene cyclases have lost γ and are αβ, others lack both β and γ and are purely α.
Scheme 1
Scheme 1
Isoprenoid biosynthesis: substrates and products
Scheme 2
Scheme 2
Carbocation mechanism for GPP, FPP, and GGPP biosynthesis
Scheme 3
Scheme 3
FPPS and GGPPS inhibitors
Scheme 4
Scheme 4
Converting FPP to cyclic products
Scheme 5
Scheme 5
Formation of diterpenes from GGPP
Scheme 6
Scheme 6
Diphosphate acts as a general base in the conversion of DMAPP to isoprene, catalyzed by isoprene synthase.
Scheme 7
Scheme 7
Formation of tuberculosinol virulence factors in M. tuberculosis.
Scheme 8
Scheme 8
Formation of HMBPP: substrate, product, and possible reactive intermediates

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