title: Monitoring processivity and length control of a carbohydrate polymerase.
abstract: Carbohydrate polymerases are abundant in nature. Although they play vital physiological roles, the molecular mechanisms that they use for the controlled assembly of polymers are largely unknown. One fundamental issue is whether an enzyme utilizes a processive or distributive mechanism for chain elongation. The shortage of mechanistic information on polysaccharide-generating glycosyltransferases became apparent when we sought to carry out investigations of GlfT2, a glycosyltransferase essential for cell wall biosynthesis in Mycobacterium tuberculosis. GlfT2 catalyzes the formation of the cell wall galactan, which is a linear polysaccharide consisting of 20-40 repeating d-galactofuranose (Galf) residues. Recombinant GlfT2 can act on synthetic acceptors to produce polymers with lengths similar to those of endogenous galactan, indicating that GlfT2 has an intrinsic ability to control polymer length. To address whether GlfT2 utilizes a processive or distributive mechanism, we developed a mass spectrometry assay. Our approach, which relies on acceptors labeled with stable isotopes, provides direct evidence that GlfT2 is a processive polymerase that maintains contact with the glycan substrate through successive monomer additions. Given this finding, we probed further the catalytic mechanism of GlfT2 to address the basis of an observed kinetic lag phase. These studies suggest that GlfT2 possesses subsites for Galf residue binding and that substrates that can fill these subsites undergo efficient processive polymerization. The presence of these subsites and the kinetic lag phase are common features of processive enzymes. We anticipate that the strategies described herein can be applied to mechanistic studies of other carbohydrate polymerization reactions.
authors: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
authors: Matthew R
authors: MR
authors: Levengood
authors: Rebecca A
authors: RA
authors: Splain
authors: Laura L
authors: LL
authors: Kiessling
mesh_terms: D005685:Galactans / Q000378:metabolism*; D005700:Galactosyltransferases / Q000378:metabolism*; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D058105:Polymerization; D013379:Substrate Specificity
chemical_list: D005685:Galactans; D005700:Galactosyltransferases