Lectin AtGAL1 interacts with high-mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate-starved Arabidopsis.

Among 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi-deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high-mannose AtPAP26-S2 glycoform with AtGAL1 (At1g78850), a Pi starvation-inducible (PSI), and Galanthus nivalis agglutinin-related (mannose-binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26-S2 to modify its enzymatic properties. Far-western immunodot blotting established that AtGAL1 readily associates with AtPAP26-S2 but not the low mannose AtPAP26-S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55-kDa AtGAL1 and AtPAP26-S2 polypeptides associate to form a 112-kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein-tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi-deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2 but not AtPAP26-S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose-rich glycans of AtPAP26-S2 and consequent positive impact on AtPAP26-S2 activity and stability.

A glycoform of the secreted purple acid phosphatase AtPAP26 co-purifies with a mannose-binding lectin (AtGAL1) upregulated by phosphate-starved Arabidopsis.

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn365 and Asn422 were modified in AtPAP26-S2 to form high-mannose glycans. A 55-kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr38 and Thr39 and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating posttranscriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.

A galactokinase-like protein from the liver fluke Fasciola hepatica.

Galactokinase catalyses the ATP-dependent phosphorylation of galactose. A galactokinase-like sequence was identified in a Fasciola hepatica EST library. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein of approximately 50 kDa. The protein is monomeric, like galactokinases from higher animals, yeasts and some bacteria. The protein has no detectable enzymatic activity with galactose or N-acetylgalactosamine as a substrate. However, it does bind to ATP. Molecular modelling predicted that the protein adopts a similar fold to galactokinase and other GHMP kinases. However, a key loop in the active site was identified which may influence the lack of activity. Sequence analysis strongly suggested that this protein (and other proteins annotated as "galactokinase" in the trematodes Schistosoma mansoni and Clonorchis sinensis) are closer to N-acetylgalactosamine kinases. No other galactokinase-like sequences appear to be present in the genomes of these three species. This raises the intriguing possibility that these (and possibly other) trematodes are unable to catabolise galactose through the Leloir pathway due to the lack of a functional galactokinase.

Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process.

A highly efficient galactokinase from Bifidobacterium infantis with broad substrate specificity.

Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned and over-expressed with a yield of over 80 mg/L cell cultures. The k(cat)/K(m) value of recombinant BiGalK toward galactose (164 s(-1) mM(-1)) is 296 times higher than that of GalK from E. coli, indicating that BiGalK is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These features make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and derivatives.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii.

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Cloning, purification, crystallization and preliminary crystallographic analysis of galactokinase from Pyrococcus furiosus.

Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222(1), with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 2.9 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.

A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.

Overexpression and purification of the galactose operon enzymes from Escherichia coli.

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically characterized by gel-filtration column chromatography and isoelectric focusing, and the Km values for their substrates were determined.

Enzyme purification by genetically attached polycysteine and polyphenylalanine affinity tails.

Two novel affinity tails, polycysteine and polyphenylalanine, have been genetically attached to galactokinase (EC 2.7.1.6) and beta-galactosidase (EC 3.2.1.23) in order to facilitate their purification. A chemically synthesized DNA linker encoding four cysteine residues was thus fused in frame with the galactokinase gene. The gene product, cysteine galactokinase, was significantly retarded on a column of thiopropyl-Sepharose. Using pulse elution, cysteine galactokinase was eluted at 10 mM DTT. Under the condition used, native galactokinase did not bind to thiopropyl-Sepharose. Homopolymer tailing was employed to prepare a phenylalanine-modified beta-galactosidase. One of the obtained genetic transformants coding for a beta-galactosidase carrying 11 phenylalanine residues at the N-terminus of the enzyme was isolated. With the aid of hydrophobic interaction chromatography the modified enzyme could be purified to homogeneity on fast protein liquid chromatography using a phenyl-Superose column.

The Philadelphia variant of galactokinase in human erythrocytes: physicochemical and catalytic properties.

The Philadelphia variant of galactokinase (GALKP) is responsible for an asymptomatic disorder of galactose metabolism. Individuals with GALKP phenotype are common among black people. They exhibit reduced galactokinase (GALK) activity in their red blood cells but normal activity in their white blood cells. We explored the biochemical characteristics of hemolysates from individuals with the GALKP phenotype and from controls. In mixed hemolysates from a control and a proband, the GALK activity measured did not suggest the presence of an inhibitor. We observed that the catalytic properties, pI and thermolability in hemolysates from controls and GALKP individuals were identical. Thus, the Philadelphia variant of galactokinase seems not to alter biochemical properties of the red blood cell enzyme. A silent amino acid substitution, or the dysfunction of a regulatory gene might be likely suggested to explain the reduced enzyme activity.

Purification of human galactokinase and evidence for its existence as a monomer form.

A procedure for preparing a highly purified galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) from human erythrocytes and placenta is described, involving DEAE-Sephacel, ammonium sulfate fractionation, gel-filtration and a subsequent chromatography step on Blue Sepharose CL-6B. The final chromatography step yields a homogeneous preparation of high specific activity. The subunit molecular weight was determined to be 38 000 for both placental and erythrocyte galactokinases. Both active preparations of the native enzyme eluted from a gel filtration column gave a molecular weight of 37 000-38 000, thus suggesting the enzyme to be present in monomeric form. The isoelectric points for both crude and their respective purified enzymes was determined to be at pH 5.7. This method for purifying human galactokinase from placenta and erythrocytes represents a significant improvement over that previously reported and contradicts past evidence for the enzyme existing as a dimer.

Galactokinase of Vicia faba seeds.

Galactokinase (EC 2.7.1.6) from the dormant seeds of Vicia faba was purified approximately 1300-fold with an 18% recovery through an eight-step procedure. The preparation showed the presence of only minor contaminations as judged by disc-gel electrophoresis. The native enzyme displayed a molecular weight of approximately 60 000 (determined by Sephadex G-100 gel-filtration) and the subunit value was 30 000. The isoelectric point of the enzyme was 5.3 and the amino acid analysis showed high percentage of acidic amino acids. The pH optimum of the enzyme was 7.3 at 25 degrees C. The relative activity for phosphorylating various monosaccharides followed the order, D-galactose greater than 2-deoxy-D-galactose greater than D-galactosamine; D-fucose, L-arabinose, L-galactose and D-glucose were not phosphorylated. Whereas ATP acted as an efficient phosphate donor, ADP, GTP and UTP were unable to act in this capacity. The Km and the V values of the substrates were determined. The metal ion requirement for the enzymic activity followed the order, Mg2+ greater than Co2+ greater than Mn2+ greater than Ni2+ greater than Ca2+. The enzymic reaction was inhibited by heavy metal ions and sulphydryl reagents indicating the participation of -SH group(s) in enzymic catalysis. Product inhibition was observed; galactose 1-phosphate and ADP were competitive and non-competitive inhibitors, respectively. Seed germination showed an increase in galactokinase level up to 24 h followed by a rapid decrease. The level of raffinose and stachyose decreased continually. The galactokinase level was found to be sufficiently high to phosphorylate the liberated galactose. No free galactose was observed at any stage of germination.

Purification and properties of galactokinase from Tetrahymena thermophila.

Galactokinase (EC 2.7.1.6; ATP: D-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50000-55000. The holoenzyme consists of two subunits of approx. 28000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1. Optimal activity was obtained at pH 7.8 and 41 degrees C, with no added monovalent salt. D-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2'-dATP and 3'-dATP as phosphate donors; ADP and adenosine-5'-[gamma-thio]triphosphate are inhibitory. The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ greater than Co2+ greater than Mn2+ greater than Fe2+. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.

Isolation, purification and partial characterization of galactokinase from Chinese hamster liver.

1. Galactokinase was purified from Chinese hamster liver. The purification process consisted of an initial biphasic partition separation followed by column chromatography on DEAE-cellulose. DEAE-Sephadex, Sephacryl S-200 and hydroxyapatite. 2. The enzyme was stabilized during the purification procedure by the inclusion of 10% glycerol, 1 mM phosphate and 20 mM beta-mercaptoethanol in all the buffer solutions. 3. Chromatography on hydroxyapatite separated two forms of galactokinase, one of which was purified to homogeneity using gel electrophoresis. 4. The purified galactokinase has a mol. wt of approximately 60,000 as determined by SDS gel electrophoresis and molecular sieving column chromatography. 5. The pH optimum is 7.4 and the Km for galactose is 1.16 x 10(-4) M.

A galactokinase of Mycobacterium sp. 279 galactose mutant.

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.

The occurrence of the Leloir pathway in non-pathogenic mycobacteria.

It has been found that saprophytic strains of mycobacteria can utilize D-galactose via the Leloir pathway which involves galactokinase, galactose-1-phosphate uridyl transferase and UDP-galactose-4-epimerase. The resulted glucose-1-phosphate is further converted by phosphoglucomutase to glucose-6-phosphate and the latter catabolized in glycolitic cycle to pyruvate. The particular enzymes of the galactose pathway have been fully separated by chromatography on a DEAE-cellulose column and some of them partially characterized.

Enzymes of galactose utilization in the rat tapeworm, Hymenolepis diminuta.

1. Crude enzyme preparations from Hymenolepis diminuta contained galactokinase, galactose 1-phosphate uridyl transferase and UDPgalactose 4-epimerase activity, although their specific activities were low. 2. Galactose 1-phosphate non-competitively inhibited galactose phosphorylation. This inhibition, together with the low specific activities of the enzymes in the pathway of galactose utilization, probably accounts for the inadequacy of galactose as a main nutritive carbohydrate for development of the worm.