title: Man o' war mutation in UDP-α-D-xylose synthase favors the abortive catalytic cycle and uncovers a latent potential for hexamer formation.
abstract: The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Walsh
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Polizzi
authors.forename: Renuka
authors.initials: R
authors.lastname: Kadirvelraj
authors.forename: Wesley W
authors.initials: WW
authors.lastname: Howard
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Wood
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics*; D009711:Nucleotides / Q000378:metabolism; D010641:Phenotype; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011509:Proteoglycans / Q000096:biosynthesis; D012996:Solutions; D014540:Uridine Diphosphate Xylose / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish
chemical_list: D050505:Mutant Proteins; D009711:Nucleotides; D011509:Proteoglycans; D012996:Solutions; D014540:Uridine Diphosphate Xylose; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase