Publications

• Roles of two conserved amino acid residues in the active site of galactose-1-phosphate uridylyltransferase: an essential serine and a nonessential cysteine.

  Geeganage S., Ling V.W., Frey P.A.

  Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of uridine 5'-diphosphate glucose (UDPGlc) and galactose-1-phosphate into uridine 5'-diphosphate galactose (UDPGal) and glucose-1-phosphate through a double displacement mechanism, with the intermediate format ... >> More

  Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of uridine 5'-diphosphate glucose (UDPGlc) and galactose-1-phosphate into uridine 5'-diphosphate galactose (UDPGal) and glucose-1-phosphate through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). The covalent linkage is a phosphoramidate formed between the UMP moiety and the His 166 N(epsilon)(2) of GalT, with His 166 N(delta1) retaining a proton throughout the catalytic cycle. Cys 160 and Ser 161 in Escherichia coli GalT are engaged in hydrogen bonding with the peripheral phosphoryl oxygen atoms of the substrate in the crystalline UMP-enzyme and in the crystalline complex of H166G-GalT with UDPGlc [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569; Thoden, J. B., Ruzicka, F. J., Frey, P. A., Rayment, I., and Holden, H. M. (1997) Biochemistry 36, 1212-1222]. Site-directed mutagenesis, thermodynamic, transient kinetic, and steady-state kinetic studies have been performed to investigate the roles of Cys 160 and Ser 161 in catalysis. The absence of the thiol group of Cys 160 in the variants C160S and C160A did not seriously alter the enzymatic activity. However, the variant S161A displayed 7000-fold less activity than wild-type GalT. The low activity of S161A was directly related to impaired uridylylation rate constant (3.7 x 10(-)(2) s(-)(1)) and de-uridylylation rate constant (0.5 x 10(-)(2) s(-)(1)) resulting from a higher kinetic barrier for uridylyl-group transfer by the variant S161A as compared with the wild-type GalT. Equilibrium uridylylation studies showed that neither Cys 160 nor Ser 161 was involved in stabilizing the uridylyl-enzyme intermediate. The results lead to the conclusion that the conserved Cys 160 does not play a critical role in catalysis. Ser 161 is most likely involved in donating a hydrogen bond to the beta-phosphoryl group of a substrate, thereby providing proper orientation for nucleophilic catalysis. << Less

  Biochemistry 39:5397-5404(2000) [PubMed] [EuropePMC]

• Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase.

  McCorvie T.J., Kopec J., Pey A.L., Fitzpatrick F., Patel D., Chalk R., Shrestha L., Yue W.W.

  Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular ... >> More

  Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants. << Less

  Hum. Mol. Genet. 25:2234-2244(2016) [PubMed] [EuropePMC]

• Uridyl transferases and the formation of uridine diphosphogalactose.

  KALCKAR H.M., BRAGANCA B., MUNCH-PETERSEN H.M.

  Nature 172:1038-1038(1953) [PubMed] [EuropePMC]

• Significance of metal ions in galactose-1-phosphate uridylyltransferase: an essential structural zinc and a nonessential structural iron.

  Geeganage S., Frey P.A.

  Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-en ... >> More

  Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mol of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 A from His 166 in active site, and the iron site lies 30 A from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that C52S-, C55S-, and H164N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1,10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential. << Less

  Biochemistry 38:13398-13406(1999) [PubMed] [EuropePMC]

• Purification and properties of galactose 1-phosphate uridyl transferase from Escherichia coli.

  KURAHASHI K., SUGIMURA A.

  J Biol Chem 235:940-946(1960) [PubMed] [EuropePMC]

• Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum.

  Nishimoto M., Kitaoka M.

  We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-ph ... >> More

  We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars. << Less

  Appl. Environ. Microbiol. 73:6444-6449(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

  Wong L.J., Sheu K.F., Lee S.L., Frey P.A.

  Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose i ... >> More

  Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates. << Less

  Biochemistry 16:1010-1016(1977) [PubMed] [EuropePMC]

• Galactose 1-phosphate uridylyltransferase of Escherichia coli. II. Further purification and characterization.

  Saito S., Ozutsumi M., Kurahashi K.

  J Biol Chem 242:2362-2368(1967) [PubMed] [EuropePMC]