Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron‐withdrawing aryl‐alkyl side chains which inhibited the growth of Gram‐negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ～1–4 μg mL^?1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially “rescued” by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (～2–6 μg mL^?1) against Gram‐positive but not Gram‐negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ～1–2 μg mL^?1.     

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 (K_i～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.     

Insight into Isoprenoid Biosynthesis by Functional Analysis of Isoprenyl Diphosphate Synthases from Mycobacterium vanbaalenii and Mycobacterium tuberculosis

Comprehensive functional analyses of E-isoprenyl diphosphate synthases (E-IDSs) from nonpathogenic Mycobacterium vanbaalenii have been performed. Mv0992 and Mv1577 represent a nonaprenyl diphosphate (E-C₄₅) synthase and a geranylgeranyl diphosphate (E-C₂₀) synthase, respectively. Although Mv3536 was identified as an E-C₂₀ synthase using a single enzyme, co-incubation of Mv3536 and Z-IDSs (Mv4662 and Mv3822) strongly suggested it releases an intermediate geranyl diphosphate (E-C₁₀) during a continuous condensation reaction. Mv0992 and Mv3536 functions differed from those of the previously reported pathogenic Mycobacterium tuberculosis homologues Rv0562 and Rv2173, respectively. Re-analysis of Rv0562 and Rv2173 demonstrated that their functions were similar to those of Mv0992 and Mv3536 (Rv0562: E-C₄₅ synthase; Rv2173: E-C_10–15 synthase). The newly proposed functions of Rv0562 and Rv2173 would be in the biosynthesis of menaquinone and glycosyl carrier lipids essential for growth. Furthermore, a reduced allylic diphosphate could be used as the Z-IDS of the Mv3822 substrate, thereby introducing a potentially novel pathway of cyclic sesquarterpene biosynthesis.     

An artificial substrate for undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26

In order to investigate the reactivity of the homologs of homoallylic substrates, we examined the enzymatic reactions of E- or Z-3-methylhex-3-enyl diphosphate with farnesyl diphosphate using the undecaprenyl diphosphate synthase (UPS) of Micrococcus luteus B-P 26. E-3-methylhex-3-enyl diphosphate reacted with farnesyl diphosphate to give a chiral (4S)-(2Z,6E,10E,14E)-4-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. However, the Z-isomer is not accepted as a substrate for the bacterial UPS at all.     

The DiPPro Approach: Synthesis,Hydrolysis, and Antiviral Activity of Lipophilic d4T Diphosphate Prodrugs

Bioreversible protection of the β‐phosphate group of nucleoside diphosphates (NDPs) as bis(acyloxybenzyl)phosphate esters is presented. To investigate the structure–activity relationship of these potential NDP prodrugs (DiPPro drugs) a series of DiPPro compounds was synthesized bearing fatty acids of various lengths and d4T as a model nucleoside. For synthesis of the lipophilically modified diphosphate group, preformed phosphoramidites were allowed to react with nucleotides, and the β‐P^III moiety was subsequently oxidized. The chemical and enzymatic stability of these prodrugs was studied in different media such as phosphate buffer (pH 7.3) or CEM cell extracts. In all media, the hydrolysis rate was clearly dependent on the acyl moiety and decreased with increasing alkyl chain length. The compounds showed a markedly lower half‐life in cell extracts than in pH 7.3 phosphate buffer due to the presence of enzyme‐catalyzed cleavage. In all media, the DiPPro compounds released d4T diphosphate (d4TDP) as the main product beside d4TMP. In antiviral assays, the compounds proved to be at least as potent as d4T against HIV‐1 and 2 in wild‐type CEM/0 cells. As a proof of concept, compounds with longer acyl residues showed very good anti‐HIV activities in thymidine‐kinase‐deficient cells (CEM/TK^?), indicating their ability to penetrate cell membranes and the delivery of phosphorylated metabolites.     

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI‐1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI‐2) is a flavoenzyme found in bacteria that is completely absent from human. IDI‐2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady‐state kinetic studies of the enzyme indicated that FMNH₂ (K_M =0.3 μM ) bound before isopentenyl diphosphate (K_M =40 μM ) in an ordered binding mechanism. An X‐ray crystal structure at 1.4 Å resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI‐2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence‐similar and structure‐related pathogens such as Enterococcus faecalis or Staphylococcus aureus.     

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.     

Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.     

A Detailed Study of Antibacterial 3‐Acyltetramic Acids and 3‐Acylpiperidine‐2,4‐diones

Inspired by the core fragment of antibacterial natural products such as streptolydigin, 3‐acyltetramic acids and 3‐acylpiperidine‐2,4‐diones have been synthesised from the core heterocycle by direct acylation with the substituted carboxylic acids using a strategy which permits ready access to a structurally diverse compound library. The antibacterial activity of these systems has been established against a panel of Gram‐positive and Gram‐negative bacteria, with activity mostly against the former, which in some cases is very potent. Data consistent with modes of action against undecaprenylpyrophosphate synthase (UPPS) and/or RNA polymerase (RNAP) for a small subset of the library has been obtained. The most active compounds have been shown to exhibit binding at known binding sites of streptolydigin and myxopyronin at UPPS and RNAP. These systems offer potential for their antibacterial activity, and further demonstrate the use of natural products as biologically validated starting points for drug discovery.     

Sesquiterpene Synthase‐Catalysed Formation of a New Medium‐Sized Cyclic Terpenoid Ether from Farnesyl Diphosphate Analogues

Terpene synthases catalyse the first step in the conversion of prenyl diphosphates to terpenoids. They act as templates for their substrates to generate a reactive conformation, from which a Mg²⁺‐dependent reaction creates a carbocation–PP_i ion pair that undergoes a series of rearrangements and (de)protonations to give the final terpene product. This tight conformational control was exploited for the (R)‐germacrene A synthase– and germacradien‐4‐ol synthase–catalysed formation of a medium‐sized cyclic terpenoid ether from substrates containing nucleophilic functional groups. Farnesyl diphosphate analogues with a 10,11‐epoxide or an allylic alcohol were efficiently converted to a 11‐membered cyclic terpenoid ether that was characterised by HRMS and NMR spectroscopic analyses. Further experiments showed that other sesquiterpene synthases, including aristolochene synthase, δ‐cadinene synthase and amorphadiene synthase, yielded this novel terpenoid from the same substrate analogues. This work illustrates the potential of terpene synthases for the efficient generation of structurally and functionally novel medium‐sized terpene ethers.     

Key Enzymes Involved in the Synthesis of Hops Phytochemical Compounds: From Structure,Functions to Applications

Humulus lupulus L. is an essential source of aroma compounds, hop bitter acids, and xanthohumol derivatives mainly exploited as flavourings in beer brewing and with demonstrated potential for the treatment of certain diseases. To acquire a comprehensive understanding of the biosynthesis of these compounds, the primary enzymes involved in the three major pathways of hops’ phytochemical composition are herein critically summarized. Hops’ phytochemical components impart bitterness, aroma, and antioxidant activity to beers. The biosynthesis pathways have been extensively studied and enzymes play essential roles in the processes. Here, we introduced the enzymes involved in the biosynthesis of hop bitter acids, monoterpenes and xanthohumol derivatives, including the branched-chain aminotransferase (BCAT), branched-chain keto-acid dehydrogenase (BCKDH), carboxyl CoA ligase (CCL), valerophenone synthase (VPS), prenyltransferase (PT), 1-deoxyxylulose-5-phosphate synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), Geranyl diphosphate synthase (GPPS), monoterpene synthase enzymes (MTS), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS_H1), chalcone isomerase (CHI)-like proteins (CHIL), and O-methyltransferase (OMT1). Furthermore, research advancements of each enzyme in terms of reaction conditions, substrate recognition, enzyme structures, and use in engineered microbes are described in depth. Hence, an extensive review of the key enzymes involved in the phytochemical compounds of hops will provide fundamentals for their applications in beer production.     

Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase

δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase (k_cat/K_M=1.4×10^?3 μm s^?1) that produces predominantly germacradien‐4‐ol in addition to 11 % δ‐cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.     

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1–geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X‐ray diffraction were carried out by employing an Escherichia coli whole‐cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)‐eremophilene synthase. The very tight binding of (+)‐eremophilene (～0.40 μM ), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1–geoA gene cluster is required for the biosynthesis of (+)‐eremophilene derivatives.     

N‐Glycosylation with Synthetic Undecaprenyl Pyrophosphate‐Linked Oligosaccharide to Oligopeptides by PglB Oligosaccharyltransferase from Campylobacter jejuni

The oligosaccharyltransferase PglB from Campylobacter jejuni catalyses the N‐glycosylation reaction with undecaprenyl‐pyrophosphate‐linked Glc₁GalNAc₅Bac₁ (Und‐PP‐Glc₁GalNAc₅Bac₁). Experiments using chemically synthesized donors coupled to fluorescently tagged peptides confirmed that biosynthetic intermediate Und‐PP‐Bac₁ and Und‐PP‐GalNAc₂Bac₁ are transferred efficiently to the Asn residue in the consensus sequence (D/E‐X′‐N‐X‐T/S, X′,X≠P). The products were analyzed in detail by tandem MS to confirm their chemical structures.     

Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.     

Antioxidant and Biological Activities of Novel Structured Monoacylglycerol Derivatives with Phenolic Acids

Novel structured monoacylglycerol (MAG)-based phenolic lipids are synthesized from11-bromoundecanoic acid, phenolic acids, and solketal. Selected phenolic acids namely 4-hydroxy benzoic, vanillic, syringic, cinnamic, p-coumaric, sinapic, 4-fluorocinnamic, 4-hydroxyphenyl acetic acid, 3-(4-hydroxyphenyl) propanoic and dihydrocaffeic acids are employed for the synthesis of ten novel MAG-based phenolic lipids. The synthesized phenolic lipids are characterized by FT-IR, NMR, and mass spectra analysis. All the compounds are evaluated for antioxidant, antimicrobial, and cytotoxic activities. MAG derivative 8g of sinapic acid exhibits excellent antioxidant activity in both DPPH assay and inhibition of lipid oxidation assay. MAG derivative 8f bearing p-coumaric acid shows good antimicrobial activity against both Gram-positive and Gram-negative bacterial strains with a minimum inhibitory concentration (MIC) value of 6.25 µm mL⁻¹. All the synthesized compounds are found to exhibit cytotoxicity against B16, DU145, and CHO cell lines, while sinapic and p-coumaric acid derivatives exhibit better activities compared to other derivatives.     

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 C₃₅ terpenes that are biosynthesized by cyclization of Z‐type C₃₅ polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C₃₅ 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 C₃₅‐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 C₅₀‐decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E‐GGPP synthase. In addition, novel cyclic C₃₅ terpenes, (14E)‐ and (14Z)‐dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C₃₅ 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 C₃₅ terpenes could reduce both E and Z prenyl residues.     

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD⁺) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD⁺ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H₂O₂) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H₂O₂) could thus be achieved when using an in situ oxygen supply through periodic external feed of H₂O₂ during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L ⁻¹) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.