Heptaprenyl Diphosphate Research Articles

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

ABSTRACTPrenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer “polyprenyl” diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis.

Combinatorial engineering for improved menaquinone-4 biosynthesis in Bacillus subtilis

Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is difficult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA, menG, and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P43 resulted in 8.1 ± 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 ± 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr, and ispD-ispF genes in MEP module with strong promoter P43 increased the titer of MK-4 to 78.1 ± 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaK1, mvaK2, mvaD, mvaS, and mvaA) resulted in 90.1 ± 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA, and menG in menaquinone pathway with strong promoter P43 increased the titer of MK-4 to 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future.

Discovery of Lipophilic Bisphosphonates That Target Bacterial Cell Wall and Quinone Biosynthesis.

We report that alkyl-substituted bisphosphonates have activity against Bacillus anthracis Sterne (0.40 μg/mL), Mycobacterium smegmatis (1.4 μg/mL), Bacillus subtilis (1.0 μg/mL), and Staphylococcus aureus (13 μg/mL). In many cases, there is no effect of serum binding, as well as low activity against a human embryonic kidney cell line. Targeting of isoprenoid biosynthesis is involved with 74 having IC50 values of ∼100 nM against heptaprenyl diphosphate synthase and 200 nM against farnesyl diphosphate synthase. B. subtilis growth inhibition was rescued by addition of farnesyl diphosphate, menaquinone-4 (MK-4), or undecaprenyl phosphate (UP), and the combination of MK-4 and UP resulted in a 25× increase in ED50, indicating targeting of both quinone and cell wall biosynthesis. Clostridioides difficile was inhibited by 74, and since this organism does not synthesize quinones, cell wall biosynthesis is the likely target. We also solved three X-ray structures of inhibitors bound to octaprenyl diphosphate and/or undecaprenyl diphosphate synthases.

Structure, Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase.

We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a "catalytic " subunit (SaHepPPS-2) having two "DDXXD" motifs and a "regulatory" subunit (SaHepPPS-1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (Ki ∼200 nm) were N-alkyl analogues of zoledronate containing ∼C6 alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially "rescued" by the addition of menaquinone-7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.

Biosynthesis of Sesterterpenes, Head-to-Tail Triterpenes, and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites.

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head-to-tail triterpenes, and sesquarterpenes. We have identified an (all-E)-isoprenyl diphosphate synthase (E-IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl-β-curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E-IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone-7, respectively.

Accumulation of heptaprenyl diphosphate sensitizes Bacillus subtilis to bacitracin: implications for the mechanism of resistance mediated by the BceAB transporter.

Heptaprenyl diphosphate (C35 -PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C35 -PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C55 -PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C35 -PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C35 -PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C35 -PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepT or by supplementation with 1,4-dihydroxy-2-naphthoate, a co-substrate with C35 -PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C35 -PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C35 -PP inhibits both BcrC (a C55 -PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C55 -PP, rather than the antibiotic across the membrane.

Insights into TIM-barrel prenyl transferase mechanisms: crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus.

Well structured: As a new triose phosphate isomerase (TIM) barrel-fold prenyl transferase, PcrB catalyzes the production of heptaprenylglyceryl phosphate from heptaprenyl diphosphate and glycerol-1-phosphate. Crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus in complex with ligands were solved, and together with site-directed mutagenesis and bioinformatics analyses, clearly reveal the catalytic mechanism of the enzyme.

Identification and characterization of a cis,trans‐mixed heptaprenyl diphosphate synthase from Arabidopsis thaliana

In eukaryotes, dolichols (C(70-120)) play indispensable roles as glycosyl carrier lipids in the biosynthesis of glycoproteins on endoplasmic reticulum. In addition to dolichols, seed plants have other types of Z,E-mixed polyisoprenoids termed ficaprenol (tri-trans,poly-cis-polyprenol, C(45-75)) and betulaprenol (di-trans,poly-cis-polyprenol, C(30-45) and C(≥70)) in abundance. However, the physiological significance of these polyprenols has not been elucidated because of limited information regarding cis-prenyltransferases (cPTs) which catalyze the formation of the structural backbone of Z,E-mixed polyisoprenoids. In the comprehensive identification and characterization of cPT homologues from Arabidopsis thaliana, AtHEPS was identified as a novel cis,trans-mixed heptaprenyl diphosphate synthase. AtHEPS heterologously expressed in Escherichia coli catalyzed the formation of C(35) polyisoprenoid as a major product, independent of the chain lengths of all-trans allylic primer substrates. Kinetic analyses revealed that farnesyl diphosphate was the most favorable for AtHEPS among the allylic substrates tested suggesting that AtHEPS was responsible for the formation of C(35) betulaprenol. AtHEPS partially suppressed the phenotypes of a yeast cPT mutant deficient in the biosynthesis of dolichols. Moreover, in A. thaliana cells, subcellular localization of AtHEPS on the endoplasmic reticulum was shown by using green fluorescent protein fused proteins. However, a cold-stress-inducible expression of AtHEPS suggested that AtHEPS and its product might function in response to abiotic stresses rather than in cell maintenance as a glycosyl carrier lipid on the endoplasmic reticulum.

Insight into C35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z‐Prenyltransferases and Identification of Dehydroheptaprenylcyclines

Nonpathogenic Mycobacterium species produce rare cyclic C(35) terpenes that are biosynthesized by cyclization of Z-type C(35) polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C(35) terpenes, we carried out functional analyses of three Z-prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z-prenyltransferase, biosynthesizes C(35)-heptaprenyl diphosphate as a main product from (E,E)-farnesyl diphosphate (E,E-FPP) and (E,E,E)-geranylgeranyl diphosphate (E,E,E-GGPP), but produces a C(50)-decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E-GGPP synthase. In addition, novel cyclic C(35) terpenes, (14E)- and (14Z)-dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C(35) terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E-FPP and E,E,E-GGPP, and the prenylreductase responsible for the biosynthesis of C(35) terpenes could reduce both E and Z prenyl residues.

Selectivity in substrate–enzyme complexation studied by surface forces measurement

The selectivity of substrate in substrate–enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM). This enzyme is composed of two dissociable subunits, which exhibits a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). We have recently succeeded to directly demonstrate a specific interaction involved in this enzyme reaction and obtain new insights into the molecular mechanism of the reaction using the approach based on the colloidal probe AFM. The AFM measurement showed the adhesive force between the subunits only in the presence of both Mg2+ and FPP. In this study, we studied the substrate selectivity in the complexation by monitoring the adhesive force. The substrates studied are pyrophosphate (PPi), isopentenyl diphosphate (IPP), geranyl diphosphate (GPP), farnesyl monophosphate (FP), and farnesyl geranyl diphosphate (FGPP). No adhesion was observed in the case of PPi, IPP, and GPP. On the other hand, the significant adhesion was observed for phosphate derivatives, which bear prenyl units longer than three. This is in good agreement with the selectivity of the substrates by this enzyme, which catalyzes the condensation reaction of four IPP molecules with FPP to give heptaprenyl (C35) diphosphates. Our study showed a useful methodology for examining the elemental processes of biological reactions.

Direct Observation of Substrate−Enzyme Complexation by Surface Forces Measurement1

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.

Direct Observation of Specific Interaction between Enzyme-substrate Complexes Using Colloidal Probe Atomic Force Microscopy

Abstract The interactions between the enzyme-substrate complex of heptaprenyl diphosphate synthase were directly investigated using colloidal probe atomic force microscopy. This enzyme is composed of two dissociable subunits which exhibit a catalytic activity only when they are associated together in the presence of Mg2+ and farnesyl diphosphate (FPP). We observed the adhesive force between the subunits only in the presence of both Mg2+ (0.1 mM) and FPP (15 μM). This is the first direct demonstration of the specific interaction involved in the enzyme reaction.

Substrate specificities of medium-prenylchain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases

In order to investigate substrate specificities of medium-prenylchain elongating enzymes, hexaprenyl diphosphate (HexPP) synthase from Micrococcus luteus B-P 26 and heptaprenyl diphosphate (HepPP) synthase from Bacillus subtilis, we examined the reactivities of 3-alkyl group homologs of allylic or homoallylic substrates for these enzymes. Only one molecule of but-3-enyl diphosphate ( 2b), which lacks a methyl group at the 3-position of isopentenyl diphosphate ( 2a) condensed with farnesyl diphosphate ( 1, FPP) to give E-norgeranylgeranyl diphosphate in the reaction catalyzed by either enzyme. 3-Ethylbut-3-enyl diphosphate ( 2c) reacted as a homoallylic substrate with FPP to give a mixture of (all- E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all- E)-3,7-diethyl-11,15,19-trimethyleicosa-2,6,10,14,18-pentaenyl diphosphates by the condensation with one or two molecules of ( 2c), respectively, by the reaction of HexPP synthase. 3-Propylbut-3-enyl diphosphate ( 2d) also reacted with FPP to give a mixture of the corresponding single and double condensation products, respectively, by the action of the same synthase. On the other hand, HepPP synthase reaction of ( 2c) or ( 2d) with FPP gave (all- E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate or (all- E)-3-propyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate as a single product, respectively. However, 3-butylbut-3-enyl diphosphate ( 2e), norfarnesyl diphosphate ( 1a) and norgeranylgeranyl diphosphate ( 1b) were never accepted as substrates by either of the enzymes at all.

Artificial substrates of medium-chain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases

We examined the reactivity of 3-alkyl group homologues of farnesyl diphosphate or isopentenyl diphosphate for medium-chain prenyl diphosphate synthases, hexaprenyl diphosphate- or heptaprenyl diphosphate synthase. But-3-enyl diphosphate, which lacks the methyl group at the 3-position of isopentenyl diphosphate, condensed only once with farnesyl diphosphate to give E-norgeranylgeranyl diphosphate by the action of either enzyme. However, norfarnesyl diphosphate was never accepted as an allylic substrate at all. 3-Ethylbut-3-enyl diphosphate also reacted with farnesyl diphosphate giving a mixture of (all- E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all- E)-3,7-diethyl-11,15,19-trimethylicosa-2,6,10,14,18-pentaenyl diphosphates by hexaprenyl diphosphate synthase. On the other hand, heptaprenyl diphosphate synthase reaction of 3-ethylbut-3-enyl diphosphate with farnesyl diphosphate gave only (all- E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate.

Chain length determination of prenyltransferases: both heteromeric subunits of medium-chain (E)-prenyl diphosphate synthase are involved in the product chain length determination.

Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638-14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate. However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50). These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme.

Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus.

A member of the medium-chain prenyl diphosphate synthases, Bacillus stearothermophilus heptaprenyl diphosphate synthase, catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce (all-E)-C35 prenyl diphosphate as the ultimate product. We previously showed that the product specificity of short-chain prenyl diphosphate synthases is regulated by the structure around the first aspartate-rich motif (FARM). The FARM is also conserved in a subunit of heptaprenyl diphosphate synthase, component II', which suggests that the structure around the FARM of component II' regulates the elongation. To determine whether component II' regulates the product chain length by a mode similar to that of the short-chain prenyl diphosphate synthases, we replaced a bulky amino acid at the eighth position before the FARM of component II', isoleucine 76, by glycine and analyzed the product specificity. The mutated enzyme, I76G, can catalyze condensations of isopentenyl diphosphate beyond the native chain length of C35. Moreover, two mutated enzymes of A79Y and S80F, which have a single replacement to the aromatic residue at the fourth or the fifth position before the FARM, mainly yielded a C20 product. These results strongly suggest that a common mechanism controls the product chain length of both short-chain and medium-chain prenyl diphosphate synthases and that, in wild-type heptaprenyl diphosphate synthase, the prenyl chain can grow on the surface of the small residues at positions 79 and 80, and the elongation is precisely blocked at the length of C35 by isoleucine 76.

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.

Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases. Formation of a hybrid-type heptaprenyl diphosphate synthase.

Among prenyltransferases that catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components. These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself. In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26. As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity. The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B. subtilis and B. stearothermophilus. There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate synthases.

Two subunits of heptaprenyl diphosphate synthase of Bacillus subtilis form a catalytically active complex.

Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon [Zhang, Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419]. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.

Identification of a Novel Gene Cluster Participating in Menaquinone (Vitamin K2) Biosynthesis: CLONING AND SEQUENCE DETERMINATION OF THE 2-HEPTAPRENYL-1,4-NAPHTHOQUINONE METHYLTRANSFERASE GENE OFBACILLUS STEAROTHERMOPHILUS

We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396-18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of S-adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes.