Methylerythritol phosphate pathway of isoprenoid biosynthesis

Abstract

Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.

Figure 1

Pathways for the biosyntheses of isoprenoid precursors (IPP and DMAPP) and their well-defined distributions among different kingdoms. A. The mevolanate pathway (MVA pathway) in animals, plants (cytosol), fungi, and archaea; B. The modified MVA pathway in Methanocaldococcus jannaschii; C. The methylerythritol phosphate pathway (MEP pathway) in eubacteria, green algae, and the plastids of higher plants.

Figure 2

A. Mechanism of TPP mediated condensation between pyruvate (10) and glyceraldehyde 3-phosphate (11) in DXS catalysis. B. The DXR retro-aldol/aldol rearrangement mechanistic model. In the retro-aldol/aldol model, a side reaction may occur through the C-C bond rotation in fragment 22 followed by the recombination between 21 and 22 to produce (3S, 4S)-DXP (23), a stereo-isomer of the DXR native substrate (3S, 4R)-DXP (12). C. Unique IspF-chemistries. Besides the native IspF catalysis (15 → 16 conversion), two more IspF chemistries (14 → 24 and 15 → 25 conversions) are discovered in some organisms.

Figure 3

X-ray crystallographic and EPR spectroscopic characterizations of IspG. A. Structure of Aquifex aeolicus IspG. IspG is a homodimer assembled in a head-to tail fashion. Each subunit has two domains, the proposed MEcPP binding N-terminal domain and the C-terminal [4Fe-4S] cluster domain. The proposed IspG active site is located at the interface between the N-terminal of one subunit and the C-terminal of the other subunit as the distance between the MEcPP binding site and the [4Fe-4S] cluster within the same subunit is too far (~56 Å). B. Pre-steady state characterizations of a thermophilic IspG using dithionite as the reductant at 55 °C. C. Pre-steady state characterizations of a thermophilic IspG using reduced methylviologen as the reductant. A single turnover at 25 °C takes ~ 10 s, which is the time period of FeS_B formation (trace C). The FeS_A species is not observed in this scenario (trace A) (adapted from (64)).

Figure 4

Proposed IspG mechanistic models. A. Symbols used to represent the iron-sulfur clusters. B. The epoxide model. According to this model, the epoxide (26) is an obligate intermediate of the MEcPP reductive dehydration. Once the epoxide (26) is formed, two sequential one-electron reductions mediated by the iron-sulfur cluster lead to HMBPP (17) formation. The Lewis acidity of the [4Fe-4S] cluster might also facilitate the dehydration process (28 → 17 conversion). C. Cation and organometallic models. In these two models, the formation of a cation intermediate (29) by the C₂ C-O cleavage is the first step. Once the cation intermediate (29) is formed, subsequent reductive dehydration can follow either the cation model via a radical cation intermediate (31) or the organometallic model via the organometallic intermediate (32). D. IspG-catalyzed reactions. IspG can catalyze both the reductive deoxygenation of 26 and the reductive dehydration of 16 to 17. However, in the absence of reductants, IspG catalyzes an irreversible 26 → 16 conversion. MEcPP (16) itself is stable for weeks at room temperature, while in the presence of holo-IspG and the absence of reductants, a positional isotopic exchange (16a↔16b) is observed.

Figure 5

Representative IspH structures revealing IspH structural flexibility. A. The crystal structure of IspH-HMBPP complex at 1.7 Å resolution. This structure reveals a few important features, including the direct coordination between the HMBPP C₄-OH to the [4Fe-4S] cluster unique iron site and a short distance (2.8 – 3.0 Å) between the HMBPP olefinic functional group (C₂ and C₃ carbons) and the [4Fe-4S] cluster unique iron site. B. The IspH-IPP complex. In this structure, the active site has two water molecules. In addition, IPP C₁ carbon adopts a conformation distinct from that in the IspH-HMBPP complex in Figure 5A. C. The IspH-DMAPP complex. This structure also shows two water molecules in the active site. D. On-beam conversion of the IspH-HMBPP complex. Relative to the structure in Figure 5A, the HMBPP C₄-OH is gone. In addition, the distance between the HMBPP olefinic functional group (C₂ and C₃ carbons) to [4Fe-4S] cluster unique iron site decreases to 2.6 – 2.8 Å. It is not yet known whether this is the product complex or an intermediate. E. The IspH E126Q mutant and HMBPP complex. Relative to the structure in Figure 5A, the HMBPP C₄-OH rotates to the other side of HMBPP olefinic functional group (C₂ and C₃ carbons) and forms an internal hydrogen bond with its terminal phosphate. In this complex, there is no water in the active site.

Figure 6

Proposed IspH mechanistic models. The formation of the IspH-HMBPP complex (33) is the only well-established step and steps after 33 are highly debated (Birch reduction model vs. Organometallic model). A. Symbols used to represent the iron-sulfur clusters. B. Birch reduction model. In this model, the IspH iron-sulfur cluster has two roles: mediating two step-wise one-electron reduction steps (33 → 34 and 35 → 36) and serving as the Lewis acid to facilitate C₄-dehydration (34 → 35). C. Organometallic model. In this model, there are also two unique features: the rotation of the HMBPP C₄-OH group away from the [4Fe-4S] cluster to form an internal H-bond (39) and an iron-sulfur cluster mediated one-step two-electron reduction (39 → 40). D. Two possible binding modes for mechanistic probe 43 (48 vs. 51).

Figure 7

Stereochemistries in IspH catalysis. A. The proposed allylic anion intermediate in IspH catalysis. B. Stereochemistries in IspH catalysis. The DMAPP C₄ methyl and IPP terminal olefinic hydrogen stereochemistries are governed by two factors, the C₃-C₄ bond rotation and the source for C₄ protonation of intermediate 36.

Figure 8

IDI-2 mechanistic model and selected mechanistic probes. A. IDI-2 mechanistic model. The flavin cofactor functions as both acid and base to complete the catalytic cycle. B. A few selected IDI-2 mechanistic probes.

Figure 9

High level microbial production of artemisinin precursors, isoprene and taxol precursors through metabolic engineering of either MVA or MEP pathway. Isoprenoid production through metabolic engineering normally involves two parts: 1) the production of a common intermediate FPP (59) by FPP synthase from IPP (1) and DMAPP (2) supplied by either the MEP or the MVA pathway (Figure 1); 2) After FPP, by introducing enzymes specific to a product of interest (e.g., Artemisinin (64), Taxol (68), or Isoprene (69)), E. coli or yeast can then be tailored by introducing the corresponding genes to produce a specific product.