title: Conservation and Covariance in Small Bacterial Phosphoglycosyltransferases Identify the Functional Catalytic Core.
abstract: Phosphoglycosyltransferases (PGTs) catalyze the transfer of a C1'-phosphosugar from a soluble sugar nucleotide diphosphate to a polyprenol phosphate. These enzymes act at the membrane interface, forming the first membrane-associated intermediates in the biosynthesis of cell-surface glycans and glycoconjugates, including glycoproteins, glycolipids, and the peptidoglycan in bacteria. PGTs vary greatly in both their membrane topologies and their substrate preferences. PGTs, such as MraY and WecA, are polytopic, while other families of uniquely prokaryotic enzymes have only a single predicted transmembrane helix. PglC, a PGT involved in the biosynthesis of N-linked glycoproteins in the enteropathogen Campylobacter jejuni, is representative of one of the structurally most simple members of the diverse family of small bacterial PGT enzymes. Herein, we apply bioinformatics and covariance-weighted distance constraints in geometry- and homology-based model building, together with mutational analysis, to investigate monotopic PGTs. The pool of 15000 sequences that are analyzed include the PglC-like enzymes, as well as sequences from two other related PGTs that contain a "PglC-like" domain embedded in their larger structures (namely, the bifunctional PglB family, typified by PglB from Neisseria gonorrheae, and WbaP-like enzymes, typified by WbaP from Salmonella enterica). Including these two subfamilies of PGTs in the analysis highlights key residues conserved across all three families of small bacterial PGTs. Mutagenesis analysis of these conserved residues provides further information about the essentiality of many of these residues in catalysis. Construction of a structural model of the cytosolic globular domain utilizing three-dimensional distance constraints, provided by conservation covariance analysis, provides additional insight into the catalytic core of these families of small bacterial PGT enzymes.
authors.affiliation: Departments of Chemistry and Biology, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Vinita
authors.initials: V
authors.lastname: Lukose
authors.affiliation: Department of Chemistry, Boston University , Boston, Massachusetts 02215, United States.|Department of Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Lingqi
authors.initials: L
authors.lastname: Luo
authors.affiliation: Department of Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Dima
authors.initials: D
authors.lastname: Kozakov
authors.affiliation: Department of Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Sandor
authors.initials: S
authors.lastname: Vajda
authors.affiliation: Department of Chemistry, Boston University , Boston, Massachusetts 02215, United States.
authors.forename: Karen N
authors.initials: KN
authors.lastname: Allen
authors.affiliation: Departments of Chemistry and Biology, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D016123:Campylobacter jejuni / Q000201:enzymology*; D020134:Catalytic Domain; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D016695:Glycosyltransferases