title: Crystal structures identify an atypical two-metal-ion mechanism for uridyltransfer in GlmU: its significance to sugar nucleotidyl transferases.
abstract: N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), exclusive to prokaryotes, is a bifunctional enzyme that synthesizes UDP-GlcNAc-an important component of the cell wall of many microorganisms. Uridyltransfer, one of the reactions it catalyzes, involves binding GlcNAc-1-P, UTP and Mg(2+) ions; however, whether one or two ions catalyze this reaction remains ambiguous. Here, we resolve this using biochemical and crystallographic studies on GlmU from Mycobacterium tuberculosis (GlmU(Mtb)) and identify a two-metal-ion mechanism (mechanism-B). In contrast to well-established two-metal mechanism (mechanism-A) for enzymes acting on nucleic acids, mechanism-B is distinct in the way the two Mg(2+) ions (Mg(2+)A and Mg(2+)B) are positioned and stabilized. Further, attempts to delineate the roles of the metal ions in substrate stabilization, nucleophile activation and transition-state stabilization are presented. Interestingly, a detailed analysis of the available structures of sugar nucleotidyl transferases (SNTs) suggests that they too would utilize mechanism-B rather than mechanism-A. Based on this, SNTs could be classified into Group-I, which employs the two-metal mechanism-B as in GlmU, and Group-II that employs a variant one-metal mechanism-B, wherein the role of Mg(2+)A is substituted by a conserved lysine. Strikingly, eukaryotic SNTs appear confined to Group-II. Recognizing these differences may be important in the design of selective inhibitors against microbial nucleotidyl transferases.
authors.affiliation: Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.
authors.forename: Pravin Kumar Ankush
authors.initials: PK
authors.lastname: Jagtap
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Verma
authors.forename: Neha
authors.initials: N
authors.lastname: Vithani
authors.forename: Vaibhav Singh
authors.initials: VS
authors.lastname: Bais
authors.forename: Balaji
authors.initials: B
authors.lastname: Prakash
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C532100:GlmU protein, Mycobacterium tuberculosis; D009097:Multienzyme Complexes; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D008274:Magnesium