Posttranslational Regulation of HMG CoA Reductase, the Rate-Limiting Enzyme in Synthesis of Cholesterol.

The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.

Undecaprenyl phosphate translocases confer conditional microbial fitness.

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2-6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

Control of the innate immune response by the mevalonate pathway.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis.

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Synergistic computational and experimental studies of a phosphoglycosyl transferase membrane/ligand ensemble.

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity.

Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

Enzymatic Synthesis of Diterpenoids from iso-GGPP†III: A Geranylgeranyl Diphosphate Analog with a Shifted Double Bond.

The analog of the diterpene precursor geranylgeranyl diphosphate with a double bond shifted from C14=C15 to C15=C16 (named iso-GGPP III) has been synthesized and enzymatically converted with six bacterial diterpene synthases; this allowed the isolation of nine unnatural diterpenes. For some of the enzyme-substrate combinations, the different reactivity implemented in the substrate analog iso-GGPP III opened reaction pathways that are not observed with natural GGPP, resulting in the formation of diterpenes with novel skeletons. A stereoselective deuteration strategy was used to assign the absolute configurations of the isolated diterpenes.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.

Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology.

BACKGROUND: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.

Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Quantification of polyprenyl diphosphates in Escherichia coli cells using high-performance liquid chromatography.

Dephosphorylation of undecaprenyl diphosphate is a crucial step in the synthesis of undecaprenyl phosphate, which is essential for cell wall synthesis. We have developed a method for the quantification of intracellular polyprenyl diphosphates, which have never before been measured directly. Polyprenyl phosphates and diphosphates prepared by chemical phosphorylation of polyprenols from Staphylococcus aureus were used to establish the conditions for fractionation by ion-exchange chromatography and high-performance liquid chromatography (HPLC). By using an elution solvent containing tetraethylammonium phosphate as an ion-pair reagent for HPLC, polyprenyl phosphate and polyprenyl diphosphate with carbon numbers from 40 to 55 could be detected as separate peaks from the reversed-phase column. This analytical method was applied to lipids extracted from Escherichia coli to determine the intracellular levels of octaprenyl phosphate, undecaprenyl phosphate, octaprenyl diphosphate, and undecaprenyl diphosphate. This is the first report of separate measurement of cellular levels of polyprenyl phosphates and polyprenyl diphosphates.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates.

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.