Enzymes related to galactose utilization inPseudomonas cepacia

Pseudomonas cepacia produced a characteristic green sheen on EMB-galactose plates owing to production of galactonic acid by the constitutive membrane-associated glucose dehydrogenase of this bacterium. Mutants isolated as glucose dehydrogenase deficient (Gcd^–) also were deficient in membrane-associated galactose dehydrogenase. A strain that formed glucose dehydrogenase at 30°C but not at 40°C was also temperature sensitive with respect to formation of galactose dehydrogenase. The Gcd^– strains still utilized galactose. A second, NAD-specific, galactose dehydrogenase (not membrane associated) along with a transport system for galactose were induced during growth on galactose and constituted an alternative pathway of conversion of galactose to galactonate. Enzymes of the De Ley-Doudoroff pathway of conversion of galactonate to pyruvate and glyceraldehyde-3-phosphate were induced during growth on galactose. Unexpectedly, growth on galactose also elicited formation of enzymes of the Entner-Doudoroff (ED) route. Furthermore, mutants blocked in the ED pathway grew poorly on galactose. One interpretation of these findings is that glyceraldehyde-3-phosphate formed from galactose via the De Ley-Doudoroff route (by cleavage of 2-keto-3-deoxy-6-phosphogalaconate) is reconverted to hexose phosphate and metabolized via the ED pathway.     

Gluconate dehydratase from the promiscuous Entner-Doudoroff pathway in Sulfolobus solfataricus

An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 degrees C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner-Doudoroff pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.     

Galactose catabolism in Caulobacter crescentus.

Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.     

In vivo assessment of 6-deoxy-6-[18F]fluoro-d-galactose as a PET tracer for studying galactose metabolism

The potential of 6-deoxy-6-[¹⁸F]fluoro-d-galactose (6-[¹⁸F]FdGal) as an in vivo tracer for studying galactose metabolism in tumors and liver was investigated. High uptake and rapid clearance of the radioactivity were observed in many organs of mice after i.v. injection of the tracer. d-Galactose loading did not affect liver uptake. Three experimental tumors showed a slightly higher uptake than other tissues, and rat brain tumor was clearly visualized by autoradiography. However, the radioactivity in tumors decreased rapidly. In the liver, a significant amount of the tracer was found in a galactonate form, while this oxidation was a minor metabolic pathway in the tumors. In both tumor and liver tissues, small amounts of the tracer were incorporated into macromolecular glycoconjugate via phosphate and uridylate forms as intermediate precursors. These results indicate that 6-[¹⁸F]FdGal is not suitable for studying galactose metabolism in vivo because of the low affinity of the tracer for the metabolism.     

The DeLey-Doudoroff Pathway of Galactose Metabolism in Azotobacter vinelandii

Azotobacter vinelandii cell extracts reduced NAD and oxidized d-galactose to galactonate that subsequently was converted to 2-keto-3-deoxy-galactonate. Further metabolism of 2-keto-3-deoxy-galactonate required the presence of ATP and resulted in the formation of pyruvate and glyceraldehyde 3-P. Radiorespirometry indicated a preferential release of CO(2) at the first carbon position of the d-galactose molecule. This suggested that Azotobacter vinelandii metabolizes d-galactose via the DeLey-Doudoroff pathway. The first enzyme of this pathway, d-galactose dehydrogenase, was partially characterized. It has a molecular weight of about 74,000 Da and an isoelectric point of 6.15. The pH optimum of the galactose dehydrogenase was about 9. The apparent K(m)s for NAD and d-galactose were 0.125 and 0.56 mM, respectively. Besides d-galactose, the active fraction of this galactose dehydrogenase also oxidized l-arabinose effectively. The electron acceptor for d-galactose or l-arabinose oxidation, NAD, could not be replaced by NADP. These substrate specificities were different from those reported in Pseudomonas saccharophila, Pseudomonas fluorescens, and Rhizobium meliloti.     

Substrate and metabolic promiscuities of d‐altronate dehydratase family proteins involved in non‐phosphorylative d‐arabinose,sugar acid,l‐galactose and l‐fucose pathways from bacteria

The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d‐ altronate dehydratase protein family, and which functions as a d‐ arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l‐ xylonate is a physiological substrate that is ultimately converted to α‐ketoglutarate via so‐called Route II of a non‐phosphorylative pathway. Several hexonates, including d‐ altronate, d‐ idonate and l‐ gluconate, which are also substrates of C785_RS13685, also significantly up‐regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d‐ or l‐ glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d‐ altronate dehydratase in a novel l‐ galactose pathway, through which l‐ galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d‐ altronate. Furthermore, this pathway completely overlapped with Route III of the non‐phosphorylative l‐ fucose pathway. The ‘substrate promiscuity’ of d‐ altronate dehydratase protein(s) is significantly expanded to ‘metabolic promiscuity’ in the d‐ arabinose, sugar acid, l‐ fucose and l‐ galactose pathways.     

Homogeneous bioluminescence assay for galactosuria: interference and kinetic analysis

Elevated galactose concentration in urine is an important clinical symptom of galactosemia and other metabolic disorders. A quantitative assay for galactose using firefly luciferase bioluminescence is presented. The assay couples the galactokinase and firefly luciferase reactions. A higher concentration of galactose present in the sample produces a faster decrease in ATP concentration, which is monitored by firefly luciferase bioluminescence. The kinetic assay is modeled and analyzed. The interference between the two reactions, the interference of certain sugars and other components in the urine, the specificity, and the optimal pH for galactokinase were studied. Calibration curves were constructed and compared with a conventional spectrophotometric assay for galactose. The bioluminescence assay is relatively fast and specific for galactose with a linear range from 1 to 20 mM galactose. The effect of other galactose metabolites (galactonate and galactitol) has also been studied.     

Induction of D-galactonate dehydratase in Mycobacterium butyricum

The induced synthesis of D-galactonate dehydratase in Mycobacterium butyricum has been studied initially after addition or removal of inductor or inhibitor. The enzyme was induced by galactonate and galactose; the system reached half-maximal effect of synthesis at 3.3 mM of galactonate. The lag of about 30 min between the addition of the inductor and the appearance of the enzyme at 37 degrees C was noted. The lag was dependent on temperature and independent of inductor concentration. After the withdrawal of the inductor the expression of a supposed galactonate dehydratase-coding messenger takes place which can be blocked by streptomycin or chloramphenicol. Both the messenger (the mean life of about 38 min) and the enzyme appeared relatively stable. The enzyme synthesis was found to be under strong catabolite repression caused by glucose and several other compounds and cyclic AMP failed to increase the enzyme synthesis or to overcome the repression. Zinc ions at concentration below 1 mM proved to have no effect on the enzyme synthesis but inhibited the enzyme itself that can be restored by EDTA.     

Galactose metabolism in Rhizobium meliloti L5-30.

Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway. However, galactose metabolism was not impaired in those mutants. We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism. Mutants in this pathway have not been previously reported for any organism.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.     

Organization and nucleotide sequence of the Streptococcus mutans galactose operon

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of -galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.     

Galactose oxidation in liver.

Rats fed a high galactose diet (40%, $\text{w}\text{w}$) for 72 h excreted more than 170 μmol of galactonic acid per day in the urine and accumulated galactonic acid in several tissues, especially liver, intestine, heart, and kidney. Rat liver microsomes incubated in the presence of 30 mm galactose produced galactonic acid. The product of the oxidation of d-galactose was identified as galactonic acid by combined gas-liquid chromatography- mass spectrometry analysis of the trimethylsilyl derivative. Optimal activity was observed at pH 8.0 and was inhibited by heavy metal ions, sulfhydryl reagents, cyanide in the absence of substrate, and various drugs. The apparent K_m for galactose was 32.9 mm and V was 160 nmol galactonic acid/4 h/mg microsomal protein. The highest galactose oxidizing specific activity was found in the microsomal fraction; specific activity in the mitochondrial fraction was one half of that in the microsomal fraction, and the soluble fraction had none. The activity was specific for d-galactose, although unidentified oxidation products of d-altrose, d-talose, and 2-deoxy-d-galactose were detected by gas chromatography. Oxidation of galactose did not require oxygen. The addition of NAD⁺ and NADP⁺ to the incubation system had little effect on the galactose oxidation; FAD and FMN inhibited the activity. Galactose-dependent formation of hydrogen peroxide was demonstrated during the incubation period. Microsomes from rats and mice contained similar galactose-oxidizing activity whereas those from bovine and chicken liver sources contained 45.3 and 5.4%, respectively. The activity was not detectable in microsomes from guinea pig liver. A liver sample, obtained at autopsy of a galactosemia patient, contained 0.18 μmol of galactonate/g suggesting that an enzyme system similar to that described herein may be responsible for the production of galactonate in human galactosemics.     

Pathway for D-galactonate catabolism in nonpathogenic mycobacteria.

D-Galactonate is catabolized in saprophytic mycobacteria to give pyruvate and glyceraldehyde-3-phosphate by a pathway that involves the sequential reactions of galactonate dehydratase, 2-keto-3-deoxy-galactonate kinase, and 6-phospho-2-keto-3-deoxy-galactonate aldolase.     

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pathway, and ultimately, increasing metabolic flux through the pathway of interest. By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway by eliminating three known negative regulators of the GAL system: Gal6, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux.     

Biochemistry and Genetics of Galactose Metabolism in Group H Streptococcus Strain Challis

Galactose-negative mutants of the group H Streptococcus strain Challis were obtained by treatment with nitrosoguanidine. Enzyme assays of extracts of these mutants revealed that 12 of the mutants were lacking one of the enzymes of the Leloir pathway. Thus, the Leloir pathway is the major means of galactose metabolism in strain Challis. In addition, uridyl diphosphate galactose pyrophosphorylase, a permease function, and at least one other function are required for the utilization of galactose. The enzymes of the Leloir pathway are induced by galactose and fucose; no compounds which act as repressors of these enzymes have been found, although the system appears to be sensitive to catabolite repression. Transformation was used to map the mutants. The genes for galactose-1-phosphate uridyl transferase and glucose-4-epimerase appear to be closely linked. Within the transferase gene, six mutations have been mapped. The permease function and the undetermined functions are not linked to the Leloir pathway.     

Galactosamine is an inducer of Gal genes in Saccharomyces cerevisiae

In yeast cells galactosamine in concentrations of 0.1 1M partially inhibits the synthesis of RNA but has little effect on the protein synthesis. In vivo and in vitro studies show that galactosamine is metabolized in yeast to UDP-N-acetylhexosamines but at a reduced rate, compared to the metabolism of galactose. The addition of galactosamine to growing yeast cells leads to the induction of the galactose pathway enzymes. Studies using different mutants in the galactose genes provide evidence that galactosamine is an inducer of the galactose structural genes in yeast. The same degree of induction of galactokinase and galactotransferase, found when galactose or galactosamine were used as inducers, supports the model of coordinated regulation in the expression of the structural genes for the galactose pathway enzymes in yeast.     

Relationship between UDP-galactose 4'-epimerase activity and galactose sensitivity in yeast

UDP-galactose 4'-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to approximately 5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to approximately 5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems.