Irena Pastuszak

Purification to Apparent Homogeneity and Properties of Pig Kidneyl-Fucose Kinase

l-Fucokinase was purified to apparent homogeneity from pig kidney cytosol. The molecular mass of the enzyme on a gel filtration column was 440 kDa, whereas on SDS gels a single protein band of 110 kDa was observed. This 110-kDa protein was labeled in a concentration-dependent manner by azido-[32P]ATP, and labeling was inhibited by cold ATP. The 110-kDa protein was subjected to endo-Lys-C digestion, and several peptides were sequenced. These showed very little similarity to other known protein sequences. The enzyme phosphorylated l-fucose using ATP to form β-l-fucose-1-P. Of many sugars tested, the only other sugar phosphorylated by the purified enzyme wasd-arabinose, at about 10% the rate ofl-fucose. Many of the properties of the enzyme were determined and are described in this paper. This enzyme is part of a salvage pathway for reutilization of l-fucose and is also a valuable biochemical tool to prepare activated l-fucose derivatives for fucosylation reactions.

GDP-l-fucose Pyrophosphorylase PURIFICATION, cDNA CLONING, AND PROPERTIES OF THE ENZYME

The enzyme that catalyzes the formation of GDP-l-fucose from GTP and β-l-fucose-1-phosphate (i.e.GDP-β-l-fucose pyrophosphorylase, GFPP) was purified about 560-fold from the cytosolic fraction of pig kidney. At this stage, there were still a number of protein bands on SDS gels, but only the 61-kDa band became specifically labeled with the photoaffinity substrate, azido-GDP-l-[32P]fucose. Several peptides from this 61-kDa band were sequenced and these sequences were used for cloning the gene. The cDNA clone yielded high levels of GFPP activity when expressed in myeloma cells and in a baculovirus system, demonstrating that the 61-kDa band is the authentic GFPP. The porcine tissue with highest specific activity for GFPP was kidney, with lung, liver, and pancreas being somewhat lower. GFPP was also found in Chinese hamster ovary, but not Madin-Darby canine kidney cells. Northern analysis showed the mRNA in human spleen, prostate, testis, ovary, small intestine, and colon. GFPP was stable at 4u2009oC in buffer containing 50 mm sucrose, with little loss of activity over a 9-day period. GTP was the best nucleoside triphosphate substrate but significant activity was also observed with ITP and to a lesser extent with ATP. The enzyme was reasonably specific for β-l-fucose-1-P, but could also utilize α-d-arabinose-1-P to produce GDP-α-d-arabinose. The product of the reaction with GTP and α-l-fucose-1-P was characterized as GDP-β-l-fucose by a variety of chemical and chromatographic methods.

Trehalose synthase converts glycogen to trehalose

Trehalose (α,α‐1,1‐glucosyl‐glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacteriumu2003smegmatis, as well as recombinant TreS produced in Escherichiau2003coli, has amylase activity in addition to the maltoseu2003↔u2003trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacteriumu2003smegmatis, and also in the recombinant enzyme produced in E.u2003coli from either the M.u2003smegmatis or the Mycobacteriumu2003tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S‐200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [3H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca2+, but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition‐state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild‐type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10‐ to 20‐fold more glycogen when grown in high concentrations (≥u20032% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.

A novel trehalase from Mycobacterium smegmatis − purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (α,α‐1,1‐glucosyl‐glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M.u2003smegmatis, and its comparison to the trehalase from M.u2003tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M.u2003smegmatis also requires Mg2+ for activity, whereas the M.u2003tuberculosis trehalase does not require Mg2+. The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71u2003kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S‐300 column, suggesting a molecular mass of about 1500u2003kDa or a multimer of 20 or more subunits. The trehalase is highly specific for α,α‐trehalose and did not hydrolyze α,β‐trelalose or β,β‐trehalose, trehalose dimycolate, or any other α‐glucoside or β‐glucoside. Attempts to obtain a trehalase‐negative mutant of M.u2003smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg2+ are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.

A 17-Amino Acid Insert Changes UDP-N-Acetylhexosamine Pyrophosphorylase Specificity from UDP-GalNAc to UDP-GlcNAc

We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147–13154). Both sugar nucleotides were synthesized at nearly the same rate, although theK m for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes inEscherichia coli. The protein isolated from theAGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2–3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.

Identification and Modification of the Uridine-binding Site of the UDP-GalNAc (GlcNAc) Pyrophosphorylase

UDP-GalNAc pyrophosphorylase (UDP-GalNAcPP; AGX1) catalyzes the synthesis of UDP-GalNAc from UTP and GalNAc-1-P. The 475-amino acid protein (57 kDa protein) also synthesizes UDP-GlcNAc at about 25% the rate of UDP-GalNAc. The cDNA for this enzyme, termed AGX1, was cloned in Escherichia coli, and expressed as an active enzyme that cross-reacted with antiserum against the original pig liver UDP-HexNAcPP. In the present study, we incubated recombinant AGX1 with N3-UDP-[32P]GlcNAc and N3-UDP-[32P]GalNAc probes to label the nucleotide-binding site. Proteolytic digestions of the labeled enzyme and analysis of the resulting peptides indicated that both photoprobes cross-linked to one 24-amino acid peptide located between residues Val216 and Glu240. Four amino acids in this peptide were found to be highly conserved among closely related enzymes, and each of these was individually modified to alanine. Mutation of Gly222 to Ala in the peptide almost completely eliminated UDP-GlcNAc and UDP-GalNAc synthesis, while mutation of Gly224 to Ala, almost completely eliminated UDP-GalNAc synthesis, but UDP-GlcNAc was only diminished by 50%. Both of these mutations also resulted in almost complete loss of the ability of the mutated proteins to cross-link N3-UDP-[32P]GlcNAc or N3-UDP-[32P]GalNAc. On the other hand, mutations of either Pro220 or Tyr227 to Ala did not greatly affect enzymatic activity, although there was some reduction in the ability of these proteins to cross-link the photoaffinity probes. We also mutated Gly111 to Ala since this amino acid was reported to be necessary for catalysis (Mio, T., Yabe, T., Arisawa, M., and Yamada-Okabe, H. (1998) J. Biol. Chem. 273, 14392–14397). The Gly111 to Ala mutant lost all enzymatic activity, but interestingly enough, this mutant protein still cross-linked the radioactive N3-UDP-GlcNAc although not nearly as well as the wild type. On the other hand, mutation of Arg115 to Ala had no affect on enzymatic activity although it also reduced the amount of cross-linking of N3-UDP-[32P]GlcNAc. These studies help to define essential amino acids at or near the nucleotide-binding site and the catalytic site, as well as peptides involved in binding and catalysis.

Kidney N-Acetylgalactosamine (GalNAc)-1-phosphate Kinase, a New Pathway of GalNAc Activation

A new enzyme that phosphorylates GalNAc at position 1 to form GalNAc-α-1P was purified ∼1275-fold from the cytosolic fraction of pig kidney, and the properties of the enzyme were determined. The kinase is quite specific for GalNAc as the phosphate acceptor and is inactive with GlcNAc, ManNAc, glucose, galactose, mannose, GalN, and GlcN. This enzyme is clearly separated from galactokinase by chromatography on phenyl-Sepharose. The GalNAc kinase has a pH optimum between 8.5 and 9.0 and requires a divalent cation in the order Mg2+ > Mn2+ > Co2+, with optimum Mg2+ concentration at ∼5 mM. The enzyme was most active with ATP as the phosphate donor, but slight activity was observed with ITP, acetyl-P, and phosphoenolpyruvate. Enzyme activity was highest in porcine and human kidney and porcine liver, but was low in most other tissues. Cultured HT-29 cells also had high activity for this kinase. The purified enzyme fraction was incubated with azido-[32P]ATP, exposed to UV light, and run on SDS gels. A 50-kDa protein was labeled, and this labeling showed saturation kinetics with increasing amounts of the probe and was inhibited by unlabeled ATP. Although the most purified GalNAc kinase preparation still had two bands that labeled with ATP, maximum labeling of the 50-kDa protein, but not the 66-kDa band, was coincident with maximum GalNAc kinase activity on a column of DEAE-Cibacron blue. On Sephacryl S-300, the native enzyme has a molecular mass of 48-51 kDa, indicating that the active kinase is a monomer. The product of the reaction was characterized as GalNAc-α-1-P by various chemical procedures.