Rhea - reaction knowledgebase

Enzymes

UniProtKB ^help_outline	2 proteins
Enzyme class ^help_outline	EC 2.4.1.288 galactofuranosylgalactofuranosylrhamnosyl-N-acetylglucosaminyl-diphospho-decaprenol beta-1,5/1,6-galactofuranosyltransferase

Reaction participants Show >> << Hide

Name ^help_outline β-D-galactofuranosyl-(1→5)-β-D-galactofuranosyl-(1→4)-α-L-rhamnosyl-(1→3)-N-acetyl-α-D-glucosaminyl-diphospho-trans,octa-cis-decaprenol Identifier CHEBI:67210 Charge -2 Formula C76H125NO26P2 InChIKey^help_outline RUVOVZNNKIXXAL-ZVNAIUDSSA-L SMILES^help_outline [H][C@]1(O[C@@H](O[C@H](CO)[C@]2([H])O[C@@H](O[C@H]3[C@H](C)O[C@@H](O[C@H]4[C@H](O)[C@@H](CO)O[C@H](OP([O-])(=O)OP([O-])(=O)OC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(/C)CCC=C(C)C)[C@@H]4NC(C)=O)[C@H](O)[C@@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)CO 2D coordinates Mol file for the small molecule Search links Involved in 3 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Name ^help_outline UDP-α-D-galactofuranose Identifier CHEBI:66915 Charge -2 Formula C15H22N2O17P2 InChIKey^help_outline ZQLQOXLUCGXKHS-SIAUPFDVSA-L SMILES^help_outline [H][C@]1(O[C@H](OP([O-])(=O)OP([O-])(=O)OC[C@H]2O[C@H]([C@H](O)[C@@H]2O)n2ccc(=O)[nH]c2=O)[C@H](O)[C@H]1O)[C@H](O)CO 2D coordinates Mol file for the small molecule Search links Involved in 5 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Name ^help_outline [β-D-galactofuranosyl-(1→5)-β-D-galactofuranosyl-(1→6)]₁₄-β-D-galactofuranosyl-(1→5)-β-D-galactofuranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)-N-acetyl-α-D-glucosaminyl-diphospho-trans,octa-cis-decaprenol Identifier CHEBI:67212 Charge -2 Formula C244H405NO166P2 InChIKey^help_outline WHUJCFISQGNUIH-SGHSUKTHSA-L SMILES^help_outline [H][C@]1(O[C@@H](O[C@H](CO)[C@]2([H])O[C@@H](OC[C@@H](O)[C@]3([H])O[C@@H](O[C@H](CO)[C@]4([H])O[C@@H](OC[C@@H](O)[C@]5([H])O[C@@H](O[C@H](CO)[C@]6([H])O[C@@H](OC[C@@H](O)[C@]7([H])O[C@@H](O[C@H](CO)[C@]8([H])O[C@@H](OC[C@@H](O)[C@]9([H])O[C@@H](O[C@H](CO)[C@]%10([H])O[C@@H](OC[C@@H](O)[C@]%11([H])O[C@@H](O[C@H](CO)[C@]%12([H])O[C@@H](OC[C@@H](O)[C@]%13([H])O[C@@H](O[C@H](CO)[C@]%14([H])O[C@@H](OC[C@@H](O)[C@]%15([H])O[C@@H](O[C@H](CO)[C@]%16([H])O[C@@H](OC[C@@H](O)[C@]%17([H])O[C@@H](O[C@H](CO)[C@]%18([H])O[C@@H](OC[C@@H](O)[C@]%19([H])O[C@@H](O[C@H](CO)[C@]%20([H])O[C@@H](OC[C@@H](O)[C@]%21([H])O[C@@H](O[C@H](CO)[C@]%22([H])O[C@@H](OC[C@@H](O)[C@]%23([H])O[C@@H](O[C@H](CO)[C@]%24([H])O[C@@H](OC[C@@H](O)[C@]%25([H])O[C@@H](O[C@H](CO)[C@]%26([H])O[C@@H](OC[C@@H](O)[C@]%27([H])O[C@@H](O[C@H](CO)[C@]%28([H])O[C@@H](OC[C@@H](O)[C@]%29([H])O[C@@H](O[C@H](CO)[C@]%30([H])O[C@@H](O[C@H]%31[C@H](C)O[C@@H](O[C@H]%32[C@H](O)[C@@H](CO)O[C@H](OP([O-])(=O)OP([O-])(=O)OC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(/C)CCC=C(C)C)[C@@H]%32NC(C)=O)[C@H](O)[C@@H]%31O)[C@H](O)[C@H]%30O)[C@H](O)[C@H]%29O)[C@H](O)[C@H]%28O)[C@H](O)[C@H]%27O)[C@H](O)[C@H]%26O)[C@H](O)[C@H]%25O)[C@H](O)[C@H]%24O)[C@H](O)[C@H]%23O)[C@H](O)[C@H]%22O)[C@H](O)[C@H]%21O)[C@H](O)[C@H]%20O)[C@H](O)[C@H]%19O)[C@H](O)[C@H]%18O)[C@H](O)[C@H]%17O)[C@H](O)[C@H]%16O)[C@H](O)[C@H]%15O)[C@H](O)[C@H]%14O)[C@H](O)[C@H]%13O)[C@H](O)[C@H]%12O)[C@H](O)[C@H]%11O)[C@H](O)[C@H]%10O)[C@H](O)[C@H]9O)[C@H](O)[C@H]8O)[C@H](O)[C@H]7O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5O)[C@H](O)[C@H]4O)[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)CO 2D coordinates Mol file for the small molecule Search links Involved in 1 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Name ^help_outline H⁺ Identifier CHEBI:15378 Charge 1 Formula H InChIKey^help_outline GPRLSGONYQIRFK-UHFFFAOYSA-N SMILES^help_outline [H+] 2D coordinates Mol file for the small molecule Search links Involved in 9,176 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Name ^help_outline UDP Identifier CHEBI:58223 Charge -3 Formula C9H11N2O12P2 InChIKey^help_outline XCCTYIAWTASOJW-XVFCMESISA-K SMILES^help_outline O[C@@H]1[C@@H](COP([O-])(=O)OP([O-])([O-])=O)O[C@H]([C@@H]1O)n1ccc(=O)[nH]c1=O 2D coordinates Mol file for the small molecule Search links Involved in 542 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule

Cross-references

	RHEA:34391	RHEA:34392	RHEA:34393	RHEA:34394
Reaction direction	undefined	left-to-right	right-to-left	bidirectional
UniProtKB ^help_outline	2 proteins
EC numbers ^help_outline	2.4.1.288
MetaCyc ^help_outline		RXN-11077

Publications

Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan.

Wheatley R.W., Zheng R.B., Richards M.R., Lowary T.L., Ng K.K.

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest ... >> More

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C(4)-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs. << Less

J. Biol. Chem. 287:28132-28143(2012) [PubMed] [EuropePMC]
A tethering mechanism for length control in a processive carbohydrate polymerization.

May J.F., Splain R.A., Brotschi C., Kiessling L.L.

Carbohydrate polymers are the most abundant organic substances on earth. Their degrees of polymerization range from tens to thousands of units, yet polymerases generate the relevant lengths without the aid of a template. To gain insight into template-independent length control, we investigated how ... >> More

Carbohydrate polymers are the most abundant organic substances on earth. Their degrees of polymerization range from tens to thousands of units, yet polymerases generate the relevant lengths without the aid of a template. To gain insight into template-independent length control, we investigated how the mycobacterial galactofuranosyl-transferase GlfT2 mediates formation of the galactan, a polymer of galactofuranose residues that is an integral part of the cell wall. We show that isolated recombinant GlfT2 can catalyze the synthesis of polymers with degrees of polymerization that are commensurate with values observed in mycobacteria, indicating that length control by GlfT2 is intrinsic. Investigations using synthetic substrates reveal that GlfT2 is processive. The data indicate that GlfT2 controls length by using a substrate tether, which is distal from the site of elongation. The strength of interaction of that tether with the polymerase influences the length of the resultant polymer. Thus, our data identify a mechanism for length control by a template-independent polymerase. << Less

Proc. Natl. Acad. Sci. U.S.A. 106:11851-11856(2009) [PubMed] [EuropePMC]
Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.

Rose N.L., Completo G.C., Lin S.J., McNeil M., Palcic M.M., Lowary T.L.

The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted ... >> More

The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents. << Less

J. Am. Chem. Soc. 128:6721-6729(2006) [PubMed] [EuropePMC]
Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase.

Rose N.L., Zheng R.B., Pearcey J., Zhou R., Completo G.C., Lowary T.L.

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential fo ... >> More

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential for mycobacterial viability and thus inhibitors of the enzymes involved in its assembly are potential drugs for the treatment of mycobacterial diseases, including tuberculosis. Only two galactofuranosyltransferases, GlfT1 and GlfT2, are responsible for the biosynthesis of the entire galactan domain of the mAG and we report here the first high-throughput assay for GlfT2. Successful implementation of the assay required the synthesis of multi-milligram amounts of the donor for the enzyme, UDP-Galf, 1, which was achieved using a chemoenzymatic approach. We also describe an improved expression system for GlfT2, which provides a larger amount of active protein for the assay. Kinetic analysis of 1 and a known trisaccharide acceptor for the enzyme, 2, have been carried out and the apparent K(m) and k(cat) values obtained for the latter are in agreement with those obtained using a previously reported radiochemical assay. The assay has been implemented in 384-well microtiter plates, which will facilitate the screening of large numbers of potential GlfT2 inhibitors, with possible utility as novel anti-TB drugs. << Less

Carbohydr. Res. 343:2130-2139(2008) [PubMed] [EuropePMC]

Enzymes

Reaction participants Show >> << Hide

Cross-references

Publications

Tetrameric structure of the GlfT2 galactofuranosyltransferase reveals a scaffold for the assembly of mycobacterial arabinogalactan.

A tethering mechanism for length control in a processive carbohydrate polymerization.

Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.

Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase.