The total synthesis of moenomycin A

Moenomycin A is the only known natural antibiotic that inhibits bacterial cell wall synthesis by binding to the transglycosylases that catalyze formation of the carbohydrate chains of peptidoglycan. We report here the total synthesis of moenomycin A using the sulfoxide glycosylation method. A newly discovered byproduct of sulfoxide reactions was isolated that resulted in substantial loss of the glycosyl acceptor. A general method to suppress this byproduct was introduced, which enabled the glycosylations to proceed efficiently. The inverse addition protocol for sulfoxide glycosylations also proved essential in constructing some of the glycosidic linkages. The synthetic route is flexible and will allow for derivatives to be constructed to further analyze moenomycin A's mechanism of action.     

A streamlined synthesis for 2,3-dihydroxyterephthalamides

[reaction: see text]. 2,3-Dihydroxyterephthalamides have been synthesized through a route that avoids the protection and deprotection of the phenol groups. The procedure allows for symmetric and unsymmetric amide linkages. This synthetic sequence significantly decreases the time and cost of preparation and increases the overall yield of this class of metal chelators.     

Unraveling the pathway of lipoic acid biosynthesis

Lipoic acid is almost universally required for aerobic metabolism. However, the mechanism for its synthesis and incorporation into proteins has remained elusive. A groundbreaking study published in the December issue of Chemistry & Biology uncovers critical features of the lipoic acid biosynthetic pathway.     

Control in the N-linked glycoprotein biosynthesis pathway

N-linked glycosylation is vital for the development and maintenance of eukaryotic cells. The individual steps of this complex process are slowly being elucidated. In this issue, the Imperiali group further dissects the mechanics of oligosaccharyl transferase using substrate analogs.     

The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach

Background

Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)₈ TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. The predicted tertiary structure of V. carchariae chitinase A has located the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245 at the exterior of the catalytic cleft. These residues are surface-exposed and presumably play an important role in chitin hydrolysis.

Results

Point mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline α-chitin and colloidal chitin, chitin binding assays demonstrated a considerable decrease for mutants W70A and Y245W, and a notable increase for S33W and W231A. When the specific hydrolyzing activity was determined, mutant W231A displayed reduced hydrolytic activity, whilst Y245W showed enhanced activity. This suggested that an alteration in the hydrolytic activity was not correlated with a change in the ability of the enzyme to bind to chitin polymer. A mutation of Trp70 to Ala caused the most severe loss in both the binding and hydrolytic activities, which suggested that it is essential for crystalline chitin binding and hydrolysis. Mutations varied neither the specific hydrolyzing activity against pNP-[GlcNAc]₂, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis.

Conclusion

Our data provide direct evidence that the binding as well as hydrolytic activities of V. carchariae chitinase A to insoluble chitin are greatly influenced by Trp70 and less influenced by Ser33. Though Trp231 and Tyr245 are involved in chitin hydrolysis, they do not play a major role in the binding process of crystalline chitin and the guidance of the chitin chain into the substrate binding cleft of the enzyme.     

Global test for metabolic pathway differences between conditions

In many metabolomics applications there is a need to compare metabolite levels between different conditions, e.g., case versus control. There exist many statistical methods to perform such comparisons but only few of these explicitly take into account the fact that metabolites are connected in pathways or modules. Such a priori information on pathway structure can alleviate problems in, e.g., testing on individual metabolite level. In gene-expression analysis, Goeman's global test is used to this extent to determine whether a group of genes has a different expression pattern under changed conditions. We examined if this test can be generalized to metabolomics data. The goal is to determine if the behavior of a group of metabolites, belonging to the same pathway, is significantly related to a particular outcome of interest, e.g., case/control or environmental conditions. The results show that the global test can indeed be used in such situations. This is illustrated with extensive intracellular metabolomics data from Escherichia coli and Saccharomyces cerevisiae under different environmental conditions.     

Toward the pathway of S. aureus WTA biosynthesis

Wall teichoic acid (WTA) contributes profoundly to the virulence of Staphylococcus aureus. The successful in vitro reconstitution of poly-ribitolphosphate WTA biosynthesis using recombinant enzymes sheds new light on WTA enzymology and paves the way for developing new antibiotics that target WTA biosynthesis, as discussed in Brown et al. in a recent issue of Chemistry & Biology.     

Generality of marine prostanoid biosynthesis by the 2-oxidopentadienylcation pathway

The prostanoid, preclavulone-A (5), which is produced by the Okinawan coral Clavularia viridis is also biosynthesized by the unrelated Caribbean coral, Pseudoplexaura porosa, indicating that the biosynthesis of 5 may be widespread in coral. The biogenesis of preclavulone-A from arachidonic acid occurs by lipoxygenation at C(8), migration of oxygen from C(8) to C(9) and ring closure, in a process (marine pathway) which contrasts sharply with the mammalian (endoperoxide) route. Preclavulone-A may be a key intermediate from which other more highly oxygenated, bioactive marine prostanoids are formed.     

Degradation and reconstruction of moenomycin A and derivatives: dissecting the function of the isoprenoid chain

Moenomycin A is the only known natural product that inhibits peptidoglycan biosynthesis by binding the bacterial transglycosylases. We describe a degradation/reconstruction route to manipulate the reducing end of moenomycin A. A comparison of the biological and enzyme inhibitory activity of moenomycin A and an analogue containing a nerol lipid in place of the natural C25 lipid chain provides insight into the role of the moenocinol unit. Our results show that a lipid chain having ten carbons in moenocinol is sufficient for enzyme inhibition, but a longer chain is required for biological acitivity, apparently because the molecule must partition into biological membranes to reach its target in bacterial cells.     

On the topological features of optimal metabolic pathway regimes

In this work, the stoichiometric metabolic network ofEscherichia coli has been formulated as a comprehensive mathematical programming model, with a view to identifying the optimal redirection of metabolic fluxes so that the yield of particular metabolites is maximized. Computation and analysis has shown that the over-production of a given metabolite at various cell growth rates is only possible for a finite ordered set of metabolic structures which, in addition, are metabolite-specific. Each regime has distinct topological features, although the actual flux values differ. Application of the model to the production of 20 amino acids on four carbon sources (glucose, glycerol, lactate, and citrate) has also indicated that, for fixed cell composition, the maximum amino acid yield decreases linearly with increasing cell growth rate. However, when the cell composition varies with cell growth rate, the amino-acid yield varies in a nonlinear manner. Medium optimization studies have also demonstrated that, of the above substrates, glucose and glycerol are the most efficient from the energetic viewpoint. Finally, model predictions are analyzed in the light of experimental data.     

Use of isotope mass probes for metabolic analysis of the jasmonate biosynthetic pathway

In order to quantitatively study the jasmonate biosynthetic pathway, we chemically synthesized a pair of isotope mass probes and established a labeling protocol. The pair of mass probes used in our work were ω-bromoacetonylpyridinium bromide (BPB) and d(5)-ω-bromoacetonylpyridinium bromide (d(5)-BPB), which contain carboxylic acid reactive groups, isotopically labeled groups and permanent positive charges. High performance liquid chromatography (HPLC) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-QTOF-MS) were used for the detection of labeled standard mixtures and plant samples. In comparison to negative mode electrospray ionization detection of unlabeled analytes, the ESI signal of reverse charge labeled compounds was shown to improve by 20- to 80-fold. Accurate relative quantification was achieved as no isotopic effects of the different isotope labeled phytohormones during RP/SCX mixed-mode liquid chromatographic separation were observed. A data analysis method was established for analyzing metabolic pathways using our labeling strategy. We then applied our method and examined the jasmonate biosynthetic pathway of rice under salt stress and the premature senescence mutant. Here we found that under salt stress conditions, rice showed up-regulation in (13S)-hydroperoxyoctadecatrienoic acid (HOPT), cis-(+)-12-oxophytodienoic acid (OPDA), 3-oxo-2-(2'-pentenyl)-cyclopentane-1-octanoic acid (OPC-8) and jasmonoyl-valine (JA-Val) levels, while α-linolenic acid (LA) and jasmonic acid (JA) showed down-regulation, and three components (HPOT, OPC-8 and JA-Val) were accumulated. The premature senescence mutant showed up-regulation in all major components of the jasmonate biosynthetic pathway with the exception of LA, and an accumulation of HPOT, OPC-6 and JA-Val. This study demonstrates that our chemical stable isotope labeling strategy can be used as a powerful tool for metabolic pathway analysis of phytohormones in plants.     

Deciphering deazapurine biosynthesis: pathway for pyrrolopyrimidine nucleosides toyocamycin and sangivamycin

Pyrrolopyrimidine nucleosides analogs, collectively referred to as deazapurines, are an important class of structurally diverse compounds found in a wide variety of biological niches. In this report, a cluster of genes from Streptomyces rimosus (ATCC 14673) involved in production of the deazapurine antibiotics sangivamycin and toyocamycin was identified. The cluster includes toyocamycin nitrile hydratase, an enzyme that catalyzes the conversion of toyocamycin to sangivamycin. In addition to this rare nitrile hydratase, the cluster encodes a GTP cyclohydrolase I, linking the biosynthesis of deazapurines to folate biosynthesis, and a set of purine salvage/biosynthesis genes, which presumably convert the guanine moiety from GTP to the adenine-like deazapurine base found in toyocamycin and sangivamycin. The gene cluster presented here could potentially serve as a model to allow identification of deazapurine biosynthetic pathways in other bacterial species.     

A revised pathway proposed for Staphylococcus aureus wall teichoic acid biosynthesis based on in vitro reconstitution of the intracellular steps

Resistance to every family of clinically used antibiotics has emerged, and there is a pressing need to explore unique antibacterial targets. Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of many Gram-positive bacteria. Because WTAs play an essential role in Staphylococcus aureus colonization and infection, the enzymes involved in WTA biosynthesis are proposed to be targets for antibiotic development. To facilitate the discovery of WTA inhibitors, we have reconstituted the intracellular steps of S. aureus WTA biosynthesis. We show that two intracellular steps in the biosynthetic pathway are different from what was proposed. The work reported here lays the foundation for the discovery and characterization of inhibitors of WTA biosynthetic enzymes to assess their potential for treating bacterial infections.     

Enzymatic reduction of ribonucleotides: biosynthesis pathway of deoxyribonucleotides

Ribonucleotide reductases are enzymes that synthesize the deoxyribonucleotides required for the replication of DNA in dividing cells. They thus have a key function for the growth of microorganisms and of all plant and animal tissues. The enzymes reduce all four purine and pyrimidine ribonucleotides (as the 5′-diphosphates or triphosphates) with direct substitution of the 2′-hydroxyl group by hydrogen. The physiological reducing agents are the mercapto groups of thioredoxins, a group of small proteins, which are regenerated from the oxidized form by NADPH-dependent thioredoxin reductases. There are two known types of ribonucleotide reductases (I and II), which catalyze hydrogen transfer with the aid of protein-bound iron ions or of 5′-deoxyadenosylcobalamin (coenzyme B₁₂); free radicals can be detected in both cases. The enzymes are regulated by effector nucleotides. There may exist a homeostatic mechanism, which guarantees the supply of DNA precursors to the cell.     

Strategy for streamlined release identity testing of chromatography media

In accordance with the US Code of Federal Regulations 21CFR 211.84 (6)(d)(1), a specific identity test must be performed for the release of chromatography media (stationary phase) before use in production of human pharmaceuticals. Due to the complexity of the physical and chemical properties of these media, i.e., variable particle morphology, insolubility, and chemical inertness, the development of specific identity tests presents a challenge. In this paper we report a new strategy for media identification that uses a combination of three relatively simple techniques: Fourier transform infrared (FT-IR) and near infrared (NIR) spectroscopy in conjunction with search libraries, and particle size distribution analysis. The methods are well established and suitable for routine application in a quality control laboratory. A hierarchical selection procedure utilizing these methods permits assignment of a unique identity for each of the chromatography media in use at a given facility, and form the basis of release tests for the media. Although this strategy was developed using specific media, the generic nature of the technology and the selection strategy proposed would permit its application to other chromatography media as well.     

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway

Nonribosomal peptide synthetases (NRPSs) are modular enzymes that use a thiotemplate mechanism to assemble the peptide backbones of structurally diverse and biologically active natural products in bacteria and fungi. Unlike these canonical multi-modular NRPSs, single-module NRPS-like enzymes, which lack the key condensation (C) domain, are rare in bacteria, and have been largely unexplored to date. Here, we report the discovery of a gene cluster (gup) encoding a NRPS-like megasynthetase through genome mining. Heterologous expression of the gup cluster led to the production of two unprecedented alkaloids, guanipiperazines A and B. The NRPS-like enzyme activates two l-tyrosine molecules, reduces them to the corresponding amino aldehydes, and forms an unstable imine product. The subsequent enzymatic reduction affords piperazine, which can be morphed by a P450 monooxygenase into a highly strained compound through C–O bond formation. Further intermolecular oxidative coupling forming the C–C or C–O bond is catalyzed by another P450 enzyme. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.

Genome mining of a NRPS-like gene cluster led to the identification of two novel alkaloids with antimicrobial activity. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.     

Possibility of a non-amino acid pathway in the biosynthesis of marine-derived oxazoles

A novel avenue for oxazoles via Beckmann rearrangement of alpha-formyl ketoxime dimethyl acetals is described that indicates the possibility of a non-amino acid biosynthetic pathway in marine natural products.     

Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.