Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB exclusively transfers to the 6-position. Moreover, whereas PimB can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM 1 ) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM 1 product of PimB. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB. Importantly, genetic evidence is provided that pimB is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac 1 PIM 2 , a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis. PIMs3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes. 4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3-6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc 2 155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimBЈ, the enzyme responsible for the tran...
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the -phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic ␣-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response, host-pathogen interactions, and tumor cell metastasis. Most of the enzymes encoded in eukaryotic/prokaryotic/archaeans genomes that are responsible for the biosynthesis and modification of glycan structures are GTs 3(1). Here we have focused in the phosphatidyl-myo-inositol mannosyltransferase A (PimA), an essential enzyme of mycobacterial growth that initiates the biosynthetic pathway of key structural elements and virulence factors of Mycobacterium tuberculosis, the phosphatidyl-myo-inositol mannosides (PIM) lipomannan and lipoarabinomannan (2-5). This amphitropic enzyme catalyzes the transfer of a Manp residue from GDPMan to the 2-position of PI to form phosphatidyl...
Background: New drugs active against persistent Mycobacterium tuberculosis are needed. Results: The fructose-1,6-bisphosphate aldolase (FBA-tb) is essential for growth of M. tuberculosis, is expressed by replicating and non-replicating bacilli, and displays plasminogen binding activity. Conclusion: FBA-tb is an essential TB enzyme that might also play a role in host/pathogen interactions. Significance: FBA-tb shows potential as a novel anti-TB therapeutic target.
We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows K i against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 10 5 ). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.
Background: Knowledge of conformational changes occurring in glycosyltransferases is limited. Results: The active site of GpgS is essentially preformed as the protein proceeds along the catalytic cycle with the nucleotide sugar -phosphate playing a central role in substrate binding. Conclusion: Conformational dynamics is a major determinant of GpgS activity.Significance: This model of action might be operational in other GT-A glycosyltransferases.
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