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![Dionex HPLC chromatography of the radioactive sugars released from mycobacterial cell wall after incubation of cultures of M. smegmatis with [1-14 C]ribose. This selective labeling of arabinose should be contrasted with that shown for labeling with [U-14 C]glucose as shown in Fig. 2. Radiolabeling, isolation of cell walls, hydrolysis, and HPLC were performed as described in Materials and Methods.](profile/Michael-Scherman/publication/14675839/figure/fig2/AS:601635789209618@1520452558759/Dionex-HPLC-chromatography-of-the-radioactive-sugars-released-from-mycobacterial-cell.png)
Dionex HPLC chromatography of the radioactive sugars released from mycobacterial cell wall after incubation of cultures of M. smegmatis with [1-14 C]ribose. This selective labeling of arabinose should be contrasted with that shown for labeling with [U-14 C]glucose as shown in Fig. 2. Radiolabeling, isolation of cell walls, hydrolysis, and HPLC were performed as described in Materials and Methods.
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Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria gro...
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... originates at position 6 or 1 of the starting radioactive glucose. This is true because dihydroxyacetone phosphate and glyceraldehyde-3-phosphate are readily interconverted, thus mak- ing label that originally originated at one or the other end of the glucose always at the 3 position in both trioses. Accordingly, both nuclides from the double-labeling experiment using [1-14 C]glucose and [6-3 H]glucose will be found at position 1 of the arabinose if the triose utilized is dihydroxyacetone phosphate or at position 5 if the triose utilized is glyceraldehyde-3-phos- phate. Hence, the label in both the arabinose and, for a con- trol, the galactose were determined by using the radiolabeled sugars formed by acid hydrolysis of cell walls doubly labeled with [1-14 C]glucose and [6-3 H]glucose. The results (Table 1) were clearly in accordance with the prediction of the arabinose skeleton being formed by the nonoxidative pentose shunt path- way, as the tritium was almost exclusively at H-5 and the 14 C was mostly at C-1. Cell wall arabinan was preferentially labeled with respect to galactan when M. smegmatis cells were incubated with [1-14 C] ribose. The conclusion that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via triose or hexose decarboxylation suggested that labeling with a pentose shunt intermediate should preferentially label cell wall arabi- nan rather than cell wall galactan. Thus, M. smegmatis cultures were incubated with [1-14 C]D-ribose in the expectation that this sugar would be taken up and converted either to Rib-5-P (a pentose shunt intermediate) by direct phosphorylation or to Ru-5-P after isomerization (4) and phosphorylation. Indeed, M. smegmatis was able to convert 14 C atoms from ribose into cell wall, and analysis of these cell walls (Fig. 4) showed that, as predicted, the label was preferentially incorporated into cell wall ...
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... Polymerization of the arabinosyl moieties is catalyzed by a number of arabinosyltransferases, for which the donor is a lipid-linked arabinosyl donor, -D-arabinosyl monophosphodecaprenol (reviewed previously in references 5 and 308). Tracer methodology applied to membrane preparation of M. smegmatis to study the origin of the arabinosyl derivatives revealed that ribulose 5-phosphate was a precursor, but that ribulose 5-phosphate was not converted directly to arabinose 5-phosphate (309). Rather, the pentosyl phosphate donor was shown to be PRPP. ...
Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.
... Interestingly sugar nucleotides of arabinose (UDP-Ara or GDP-Ara) have not been identified in mycobacteria. Instead, the carbon skeleton of the arabinosyl residues are derived from 5-phosphoribosyl-1-pyrophosphate ( pRpp), a metabolite that emerges from the nonoxidative pentose shunt pathway (Scherman et al. 1995). M. tuberculosis has a single pRpp synthetase, which is responsible for the formation of pRpp and uses both ribose 5-phosphate and ATP as its substrates (Alderwick et al. 2011). ...
The mycobacterial bacillus is encompassed by a remarkably elaborate cell wall structure. The mycolyl-arabinogalactan-peptidoglycan (mAGP) complex is essential for the viability of Mycobacterium tuberculosis and maintains a robust basal structure supporting the upper "myco-membrane." M. tuberculosis peptidoglycan, although appearing to be unexceptional at first glance, contains a number of unique molecular subtleties that become particularly important as the TB-bacilli enters into nonreplicative growth during dormancy. Arabinogalactan, a highly branched polysaccharide, serves to connect peptidoglycan with the outer mycolic acid layer, and a variety of unique glycolsyltransferases are used for its assembly. In this review, we shall explore the microbial chemistry of this unique heteropolysacchride, examine the molecular genetics that underpins its fabrication, and discuss how the essential biosynthetic process might be exploited for the development of future anti-TB chemotherapies.
Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
... essential cell envelope glycoconjugates, AG and lipoarabinomannan (LAM) (Wolucka et al., 1994). It is synthesized from 5-phosphoribose-1-pyrophosphate (Scherman et al., 1995(Scherman et al., ,1996 -the product of the phosphoribosyl-pyrophosphate synthetase PrsA (Alderwick et al., 2011a) -through four reaction steps involving the Dec-P 5-phosphoribosyltransferase, UbiA (Rv3806c; Huang et al., 2005Huang et al., , 2008Alderwick et al., 2005), the phosphoribosyl-monophosphodecaprenol phosphatase Rv3807c (Jiang et al., 2011), and DprE1 (Rv3790) and DprE2 (Rv3791) responsible for the epimerization of decaprenyl-phosphate ribose to Dec-P-Ara (Mikušová et al., 2005). The AraTs involved in the formation of the arabinan domain of AG identified to date include AftA (Rv3792), responsible for the transfer of the very first Araf residues to the galactan domain of AG (Alderwick et al., 2006a), the terminal b À (1,2)-capping AraT AftB (Rv3805c; Seidel et al., 2007), AftC (Rv2673) involved in the internal a-(1,3)-branching of AG (Birch et al., 2008) and the EmbA (Rv3794) and EmbB (Rv3795) proteins involved in the formation of the Ara 6 motif of AG (Escuyer et al., 2001;Khasnobis et al., 2006). ...
Abstract Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of the most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last 10 years in the discovery and development of novel inhibitors targeting their biogenesis.
... This unique nature leads to the prediction that the enzymes that synthesize this structure should yield a number of potential drug targets [2, 3]. The D-arabinofuran residues (Araf) and D-galactofuran residues (Galf) are added to form the mature lipid-linked AG, and the immediate donor of the polymerized Araf is decaprenylphosphoryl-D-arabinose (DPA) contributing to AG and lipoarabinomannan (LAM) assembly345. Many of the enzymes catalyzing the synthesis of DPA serving as an arabinose donor in the cell wall biosynthesis have been identified [6] and utilized as the targets for the development of new drugs against TB [7, 8]. ...
Decaprenylphosphoryl-d-arabinofuranosyl (DPA), the immediate donor for the polymerized d-Araf residues of mycobacterial arabinan, is synthesized from 5-phosphoribose-1-diphosphate (PRPP) in three-step reactions. (i) PRPP is transferred to decaprenyl-phosphate (DP) to form decaprenylphosphoryl-d-5-phosphoribose (DPPR). (ii) DPPR is dephosphorylated to form decaprenylphosphoryl-d-ribose (DPR). (iii) DPR is formed to DPA by the epimerase. Mycobacterium tuberculosis Rv3806c and heteromeric Rv3790/Rv3791 have been identified as the PRPP: decaprenyl-phosphate 5-phosphoribosyltransferase and the epimerase respectively. Rv3807c, however, as the candidate of phospholipid phosphatase, catalyzing the biosynthesis of decapreny-l-phosphoryl-ribose (DPR) from decaprenylphosphoryl-β-d-5-phosphoribose by dephosphorylating, has no direct experimental evidence of its essentiality in any species of mycobacterium. In this study, Rv3807c gene was amplified from the genome of M. tuberculosis H37Rv by PCR, and was successfully expressed in Escherichia coli BL21 (DE3) via the recombinant plasmid pColdII-Rv3807c. The resulting protein with the 6× His-tag was identified by SDS-PAGE and Western blotting. The protein was predicted through bioinformatics to contain three transmembrane domains, the N-terminal peptide, and a core structure with phosphatidic acid phosphatase type2/haloperoxidase. This study provides biochemical and bioinformatics evidence for the importance of Rv3807c in mycobacteria, and further functional studies will be conducted for validating Rv3807c as a promising phospholipid phosphatase in the synthetic pathway of DPA.
... In the mid 1990s McNeil and his colleagues at Colorado State University initiated a series of important metabolic studies leading to a final definition of the DPA pathway. One of the milestones towards this goal was the implication of the non-oxidative pathway of the pentose shunt as the metabolic origin of the arabinosyl residues [108]. The authors found that incorporation of radioactive [U14 C]-glucose, [6-3 H]-glucose, [6-14 C]-glucose, [1-14 C]-glucose into the cell wall arabinans was comparable, which precluded removal of carbon by oxidation either in the oxidative branch of the pentose pathway, or by C-6 decarboxylation of UDP-glucose described for other pentoses [109,110]. ...
... The authors found that incorporation of radioactive [U14 C]-glucose, [6-3 H]-glucose, [6-14 C]-glucose, [1-14 C]-glucose into the cell wall arabinans was comparable, which precluded removal of carbon by oxidation either in the oxidative branch of the pentose pathway, or by C-6 decarboxylation of UDP-glucose described for other pentoses [109,110]. At the same time, none of the enzymatic activities leading to production of arabinose-5-phosphate from the pentose shunt product ribulose-5-phosphate, known from Gram negative organisms, were detected [108]. The authors hypothesized that an unusual pathway including 5-phosphoribose pyrophosphate (PRPP) may play a role in the biosynthesis of arabinose, which they, indeed, proved in their following study [111]. ...
Several groups working in the field of the development of new antituberculotics have recently reported active compounds targeting mycobacterial enzyme DprE1. Along with its counterpart, DprE2, it catalyses a unique epimerization reaction resulting in the synthesis of decaprenylphosphoryl arabinose, a single donor of arabinosyl residues for the build-up of arabinans, fundamental components of the mycobacterial cell wall. This review presents the historical background leading to the discovery of DprE1, focusing on the biochemical and structural characterization of this important emerging target and introducing the molecules acting on DprE1 including the development of the most successful series - the benzothiazinones, currently in late pre-clinical development, which turned to be suicide inhibitors of DprE1.
... contains lipoarabinomannan (LAM), a complex lipid glycoprotein anchored to the cell membrane by phosphatidylinositol which has structural and functional similarity to LPS, including the presence of anionic phosphate groups (Zhang et al., 1994). Biosynthesis of LAM is known to be a target for several antituberculosis agents, including the first line antitubercular agent, ethambutol (17; Fig. 4) (Scherman et al. 1995;Heijenoort, 2001). For the synthesis of the most active phenylacrylamide derivative (28; Scheme 3), the required guanylhydrazone (25a) was prepared by the microwave-assisted reaction of 3,4-dimethoxy benzaldehyde (25) with guanylhydrazine hydrochloride (26). ...
... Biosynthesis of LAM is known to be a target for several antituberculosis agents, including the first line antitubercular agent, ethambutol (17;Fig. 4) (Scherman et al. 1995; Heijenoort, 2001). For the synthesis of the most active phenylacrylamide derivative (28; Scheme 3), the required guanylhydrazone (25a) was prepared by the microwave-assisted reaction of 3,4-dimethoxy benzaldehyde (25) with guanylhydrazine hydrochloride (26). ...
... It is therefore considered that pRpp is required at all times during the life cycle of both prokaryotic and eukaryotic cells (Hove-Jensen 1989; Tozzi et al. 2006). In addition to these central metabolic processes, members belonging to the Corynebacteriacae, such as mycobacteria, have evolved an almost unique biochemical pathway, utilizing pRpp as a high-energy biosynthetic precursor for cell wall arabinan biosynthesis (Wojtkiewicz et al. 1988; Scherman et al. 1995 Scherman et al. , 1996 Figure 1). The gene product of Rv3806c encodes for a membrane bound pRpp:decaprenol-1- monophosphate 5-phosphoribosyltransferase (DPPR synthase) which catalyzes the formation of DPPR and pyrophosphate from pRpp and decaprenol-1-monophosphate (DP; Huang et al. 2005). ...
... Evidence suggesting that Mt-prsA is an essential gene for the maintenance of cellular integrity Mt-prsA is predicted to be an essential gene as determined by high-density transposon mutagenesis (Sassetti et al. 2003). This is not unsurprising, considering the apparent physiological importance of Mt-PrsA in the provision of pRpp for central metabolic pathways and the formation of the cell wall biosynthetic precursor DPA (Wolucka et al. 1994; Scherman et al. 1995 ). We have previously used Corynebacterium glutamicum as an excellent model system for the study of complex cell wall biosynthetic processes, and we have reported the phenotypic characterization of several cell wall mutants, which would otherwise be essential in mycobacterial systems (Gande et al. 2004; Alderwick et al. 2005; Alderwick, Dover, et al. 2006; Seidel et al. 2007; Birch et al. 2008). ...
... Interestingly, the homology model of Mt-PrsA suggests that a salt bridge between the equivalent residues does not occur thus reducing the stability of the dimer interface. In mycobacteria, the biosynthesis of pRpp is required at a rate which is sufficient to not only supply central metabolic processes, such as de novo nucleoside biosynthesis, but also at a necessary level to meet the increased demands for pRpp during cell wall biosynthesis (Scherman et al. 1995Scherman et al. , 1996). It seems plausible that pRpp synthetases from Corynebacterineae species have evolved a highly efficient version of the enzyme to account for the increased metabolic flux of pRpp, during various physiological states. ...
Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan
to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination
of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The
source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate
(pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of
pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical
study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase.
... Biosynthesis of DPA from 5-phosphoribopyrophosphate (pRpp) is performed by three enzymes: Rv3806c, Rv3790, Rv3791. 63,64 First, the decaprenylphosphoryl-5-phosphoribose (DPPR) synthase Rv3806c, identified as UbiA in C. glutamicum, 65 transfers pRpp to decaprenyl phosphate to form DPPR, 66 then the 5 0 -phosphate is removed to give decaprenylphosphoryl ribose (DPR) by a phosphatase, putatively Rv3807c, 67 and DPR is then epimerized to DPA. The epimerization was initially proposed to occur via an oxidation-reduction process involving two enzymes (Rv3790 and Rv3791); 68 Rv3790 (or DprE 1 ) functions as decaprenylphosphoryl-b-D-ribose oxidase and Rv3791 (or DprE 2 ) as decaprenylphosphoryl-D-2-keto erythropentose reductase. ...
Drug-resistant forms of Mycobacterium tuberculosis (M. tuberculosis) are increasing worldwide, underscoring the need to develop new drugs to treat the disease. One of the factors that make tuberculosis difficult to treat is the unique architecture of the mycobacterial cell wall. In this review, we catalogue the enzymes involved in the synthesis of the mycolylarabinogalactan (mAG), a key structural component of the mycobacterial cell wall. In addition, we review the enzymes required for the synthesis of the related lipoarabinomannan (LAM), a structure that possesses immunomodulatory properties. The integrity of the mAG and LAM is critical to the viability of mycobacteria, and many of the established antimycobacterial agents target enzymes critical to the synthesis of the mAG and LAM. Recently, new enzymes catalyzing synthetic steps in the synthesis of the mAG and LAM have been characterized and their substrate specificity determined. In this report, we review recent efforts to characterize the enzymes involved in mAG and LAM synthesis and describe the compounds used to inhibit the enzymes or characterize their catalytic activity.
... Polyprenol-monophosphoryl-β-D-arabinose (DPA) is the only known Araf donor in mycobacteria (Wolucka et al., 1994). It is synthesized from 5-phosphoribose-pyrophosphate (Scherman et al., 1995; Scherman et al., 1996) following four catalytic steps which have been characterized (Huang et al., 2005; Mikušová et al., 2005; Huang et al., 2008). Since DPA is the only known Araf donor, it is expected that the arabinosylation of AG and LAM is catalyzed by membrane-associated polyprenyl-dependent glycosyltransferases (GTs) on the periplasmic side of the plasma membrane (Berg et al., 2007). ...
The re-emergence of tuberculosis in its present-day manifestations - single, multiple and extensive drug-resistant forms and as HIV-TB coinfections - has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moieties of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel who have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.
