|ProGT Pathway||Organism Information ||Organism Name||Mycobacterium tuberculosis H37Rv ATCC 25618 ||Clinical Implication||Pathogenic||Domain||Bacteria||Phylum||Actinobacteria||Classification||Family: Mycobacteriaceae|
Division or phylum: "Actinobacteria"
|Taxonomic ID (NCBI)||83332||Genome Sequence(s) ||Gene Bank||NC_000962.3 ||EMBL||AL123456||Gene Information ||NCBI Gene ID||887882||Protein information ||UniProtKB/ SwissProt ID||P9WN05||NCBI Ref Seq||NP_215518.1 ||UniProtKB Sequence||>sp|P9WN05|PMT_MYCTU Probable dolichyl-phosphate-mannose--protein mannosyltransferase OS=Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) GN=pmt PE=1 SV=2
||EMBL CDS||CCP43752.1 ||Sequence length||503 AA||Subcellular Location||Membrane||Function in Native Organism ||String||83332.Rv1002c. ||Potential Application||Rv1002c-dependent protein O-mannosylation is required for Mtb intracellular persistence and proliferation and for full virulence in SCID mice.||Additional Information||1) The attenuation of the mutant virulence is related to the inactivation of Rv1002c gene coding for the Mtb PMT. |
2) Rv1002c, a M. tuberculosis membrane protein homolog of eukaryotic protein mannosyltransferases and presented a similar hydropathy profile with PMTs of S. cerevisiae , was shown to catalyze the initial step of protein mannosylation. Bioinformatic analyses shows 11 transmembrane domains in Rv1002c and the presence of the conserved PMT active site residues R39, D55, E56, and R106.
|Glycosylation Recation ||Glycosylation Type||O- (Ser/Thr) linked ||Mechanism of Catalysis||Inverting||Basis of catalytic mechnism||Catalysis annotation based on experimental evidence.||CAZY Family||GT39 ||EC Number (BRENDA)||2.4.1.- ||Donor Type||Lipid linked sugars|| Donor Specificity||Polyprenol phosphate -Mannose||Accessory GT ID||ProGT10.1 || Glycan Information ||Glycan transferred||Monosaccharide (Mannose) ||Method of Glycan Indentification||Liquid chromatography-electrospray ionization-tandem mass spectrometry and affinty chromatography ConA|| Genetic evidence of Glycosyltransferase activity ||Experimental_strategies||Effect of glycosylation on acceptor protein was analyzed by Gel Electrophoresis and confirmed by mass spectrometry. ||Recombination Host/ Vector used||Vector: pSC-B (for knock out) ||Complementation Host/Vector used||vector: pMV361 || Bio Chemical Evidence of Glycosyltransferase Activity || Method of In vitro GT Assay||2 mg protein suspended in assay buffer (50 mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM DTT) to which 1.9 nmol [14C] GDP-mannose with a specific activity of 260 mCi/mmol and 1mmol of the synthetic peptide (AAAPPAPATPVAPPPPHHHHHH) were added. The final reaction volume was brought to 500 µL with assay buffer. The assay mixture was incubated at 37°C for 30 min and the enzymatic reaction was stopped by heating at 85°C for 2 min. Suspension centrifuge to pellet particulate material, and the supernatant recovered. Radiolabeled peptides were purified from the supernatant of the enzymatic reaction by Nickel affinity chromatography. Each fraction was added to 10 ml of EcoLume scintillation fluid and counted on a Beckman LS6500 Scintillation counter. ||Recombination Host and vector used in vitro GT assay||Rv1002c cloned in pMH29 creating pBV90 and electroporated into M. tuberculosis H37Rv and M. smegmatis mc2 155 ||Mutantation in recombinant GT||1) wild-type Rv1002c and three mutated forms of this gene conferring A55A56, A55E56, or D55A56 substitutions in the invariant D55E56 motif were overexpressed in Mycobacterium smegmatis, and membrane preparations were assayed for in vitro PMT activity. Overexpressed wild-type Mtb Rv1002c significantly increased (66.8%) PMT activity of the M. smegmatis membranes above that observed in the M. smegmatis vector control |
2) D55E56 to A55E56 substitution resulted in decreased (56.0%) PMT activity compared with the M. smegmatis vector control.
|Acceptor Subtrate Information ||Acceptor Substrate name ||Alanine and proline-rich secreted protein Apa (50/55-kDa or 45 kDa MPT 32)||ProGPdb ID ||ProGP51||Experimental Validation of Acceptor||In vitro and in vivo||Organism ||Mycobacterium tuberculosis H37Rv||Acceptor Substrate name ||SodC||ProGPdb ID ||ProGP190||Experimental Validation of Acceptor||In vivo||Litrature ||Year Of Validation||2005 ||Reference ||Brian C. VanderVen, Jeffery D. Harder, Dean C. Crick, John T. Belisle (2005) Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways. Science. 2005 Aug 5;309(5736):941-3.||Authors ||Brian C. VanderVen, Jeffery D. Harder, Dean C. Crick, John T. Belisle||Research groups||Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.||Corresponding Author||John T. Belisle||Contacts||Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.||Reference ||Liu CF, Tonini L, Malaga W, Beau M, Stella A, Bouyssié D, Jackson MC, Nigou J, Puzo G, Guilhot C, Burlet-Schiltz O, Rivière M(2013). Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis. ||Authors ||Liu CF, Tonini L, Malaga W, Beau M, Stella A, Bouyssié D, Jackson MC, Nigou J, Puzo G, Guilhot C, Burlet-Schiltz O, Rivière M||Research groups||Scientific Research National Center, Institute of Pharmacology and Structural Biology, F-31077 Toulouse, France.||Corresponding Author||Rivière M.||Contacts||Scientific Research National Center, Institute of Pharmacology and Structural Biology, F-31077 Toulouse, France.||Reference ||Córdova-Dávalos LE, Espitia C, González-Cerón G, Arreguín-Espinosa R, Soberón-Chávez G, Servín-González L.(2014). Lipoprotein N -acyl transferase (Lnt1) is dispensable for protein O -mannosylation by Streptomyces coelicolor, 350, 72-82. https://doi.org/10.1111/1574-6968.12298||Authors ||Córdova-Dávalos LE, Espitia C, González-Cerón G, Arreguín-Espinosa R, Soberón-Chávez G, Servín-González L||Research groups||Department of Molecular Biology and Biotechnology, Institute of Biomedical Research, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, Mexico City.||Corresponding Author||Servín-González L||Contacts||Department of Molecular Biology and Biotechnology, Institute of Biomedical Research, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, Mexico City.|