The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB ؊ mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA ؊ and embB ؊ mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA ؊ and embB

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

pathogenesis ( 4-6 ). The abundance and biological importance of the Mtb lipids has resulted in extensive and elegant studies to elucidate their structures and functions ( 1-3 ). In many cases, the lipids of Mtb are unique to this pathogen or shared only with other members of this genus.Earlier studies demonstrate variability in lipid profi les among different strains of Mtb ( 7-11 ) and that minor variations in the structure of individual lipids can occur with changes in the growth environment (12)(13)(14)(15)(16)(17)(18)(19)(20). However, targeted and nontargeted assays that monitor changes in Mtb lipid profi les are generally performed by traditional TLC-based methods ( 21 ), and global lipidomics analyses in Mtb have been restricted due to limits in the technology to detect and rapidly identify a large number of lipids in a single experiment. Two-dimensional NMR was recently applied to examine global mycobacterial lipid profi les, and this approach allowed for the identifi cation of key lipid differences in 13 C-enriched cellular extracts ( 22 ). Although this approach easily detects changes in lipid patterns, it is limited by the complexity of the NMR spectra and the overlapping chemical properties of many lipids. Alternatively, MS-based lipidomic strategies allowing simultaneous detection, identifi cation, and quantifi cation of structurally diverse lipid components of Mtb also were evaluated. Leavell and Leary ( 23 ) developed an algorithm to analyze high-resolution Fourier transform-ion cyclotron Abstract The cellular envelope of Mycobacterium tuberculosis is highly distinctive and harbors a wealth of unique lipids possessing diverse structural and biological properties. However, the ability to conduct global analyses on the full complement of M. tuberculosis lipids has been missing from the repertoire of tools applied to the study of this important pathogen. We have established methods to detect and identify lipids from all major M. tuberculosis lipid classes through LC/MS lipid profi ling. This methodology is based on efficient chromatographic separation and automated ion identifi cation through accurate mass determination and searching of a newly created database ( Mtb LipidDB) that contains 2,512 lipid entities. We demonstrate the sensitive detection of molecules representing all known classes of M. tuberculosis lipids from a single crude extract. We also demonstrate the ability of this methodology to identify changes in lipid content in response to cellular growth phases. This work provides a customizable framework and resource to facilitate future studies on mycobacterial lipid biosynthesis and

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5␣-acetoxy-10␤-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10␤-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10␤-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10␤-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.Taxol biosynthesis ͉ paclitaxel ͉ taxadien-5␣-yl acetate ͉ taxadien-5␣-acetoxy-10␤-ol ͉ Taxus cuspidata T he complex diterpenoid Taxol § (paclitaxel) (1) is a potent antimitotic agent with excellent activity against a range of cancers (2). The only source of the drug, and of its precursors for semisynthetic preparation (3-5), is yew (Taxus) species (6). The supply of Taxol has been limited since the discovery of this natural product (6), and, with increasing applications in chemotherapy, the availability and cost of the drug will remain important issues (7). For the foreseeable future, the supply of Taxol and its synthetically useful precursors must continue to rely on biological methods of production, either in Taxus species or, potentially, by cell cultures derived from these plants (7). Improving the biological production yields of the drug depends on a detailed understanding of the biosynthetic pathway, the enzymes catalyzing the sequence of reactions (especially the slow steps), and the genes encoding these enzymes.The biosynthesis of Taxol (Fig. 1) involves the initial cyclization of geranylgeranyl diphosphate, the universal precursor of diterpenoids, to taxa-4(5),11(12)-diene (8), followed by extensive oxidative modification of this parent olefin and the elaboration of side-chains (9-11), although the precise order of intermediates of the pathway is largely unknown. The dearth of naturally occurring, lightly functionalized taxoids (i.e., taxoids bearing up to three oxygen functional groups) (12) provides no assistance in deciphering the early oxy...

The location and interactions of solutes in microheterogeneous environments, such as reverse micelles, critically influence understanding of many phenomena that utilize probe molecules to characterize properties in chemical, biological, and physical systems. The information gained in such studies depends substantially on the location of the probe used. Often, intuition leads to the assumption that ionic probe molecules reside in the polar water pool of a system. In this work, the location of a charged polar transition metal coordination complex in a reverse micellar system is determined using NMR spectroscopy. Despite the expected Coulomb repulsion between the surfactant headgroups and the negatively charged complex, the complex spends significant time penetrating into the hydrophobic portion of the reverse micellar interface. These results challenge the assumption that ionic probe molecules reside solvated by water in microheterogeneous environments and suggest that probe molecule location be carefully considered before interpreting data from similar systems.

CpXRh(III)-catalyzed C-H functionalization reactions are a proven method for the efficient assembly of small molecules. However, rationalization of the effects of cyclopentadienyl (CpX) ligand structure on reaction rate and selectivity has been viewed as a black box, and a truly systematic study is lacking. Consequently, predicting the outcomes of these reactions is challenging because subtle variations in ligand structure can cause notable changes in reaction behavior. A predictive tool is, nonetheless, of considerable value to the community as it would greatly accelerate reaction development. Designing a data set in which the steric and electronic properties of the CpXRh(III) catalysts were systematically varied allowed us to apply multivariate linear regression algorithms to establish correlations between these catalyst-based descriptors and the regio-, diastereoselectivity, and rate of model reactions. This, in turn, led to the development of quantitative predictive models that describe catalyst performance. Our newly-described cone angles and Sterimol parameters for CpX ligands served as highly correlative steric descriptors in the regression models. Through rational design of training and validation sets, key diastereoselectivity outliers were identified. Computations reveal the origins of the outstanding stereoinduction displayed by these outliers. The results are consistent with partial η5−η3 ligand slippage that occurs in the transition state of the selectivity-determining step. In addition to the instructive value of our study, we believe that the insights gained are transposable to other Group 9 transition metals and pave the way toward rational design of C-H functionalization catalysts.

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10␤-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperdabaculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5␣-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10␤-hydroxylase) as a taxane 13␣-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.paclitaxel ͉ taxa-4(20),11(12)-dien-5␣,13␣-diol ͉ Taxus cuspidata ͉ yew

Inorganic nanoparticles, stabilized by a passivating layer of organic molecules, form a versatile class of nanostructured materials with potential applications in material chemistry, nanoscale physics, nanomedicine and structural biology. While the structure of the nanoparticle core is often known to atomic precision, gaining precise structural and dynamical information on the organic layer poses a major challenge. Here we report a full assignment of 1H and 13C NMR shifts to all ligands of a water-soluble, atomically precise, 102-atom gold nanoparticle stabilized by 44 para-mercaptobenzoic acid ligands in solution, by using a combination of multidimensional NMR methods, density functional theory calculations and molecular dynamics simulations. Molecular dynamics simulations augment the data by giving information about the ligand disorder and visualization of possible distinct ligand conformations of the most dynamic ligands. The method demonstrated here opens a way to controllable strategies for functionalization of ligated nanoparticles for applications.