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PubMed1983

Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is representative of a large group of pseudomonad bacteria that possess coexisting alternative pathways to L-phenylalanine (as well as to L-tyrosine). These multiple flow routes to aromatic end products apparently account for the inordinate resistance of P. aeruginosa to end product analogs. Manipulation of carbon source nutrition produced a physiological state of sensitivity to p-fluorophenylalanine and m-fluorophenylalanine, each a specific antimetabolite of L-phenylalanine. Analog-resistant mutants obtained fell into two classes. One type lacked feedback sensitivity of prephenate dehydratase and was the most dramatic excretor of L-phenylalanine. The presence of L-tyrosine curbed phenylalanine excretion to one-third, a finding explained by potent early-pathway regulation of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase-Tyr (a DAHP synthase subject to allosteric inhibition by L-tyrosine). The second class of regulatory mutants possessed a completely feedback-resistant DAHP synthase-Tyr, the major species (greater than 90%) of two isozymes. Deregulation of DAHP synthase-Tyr resulted in the escape of most chorismate molecules produced into an unregulated overflow route consisting of chorismate mutase (monofunctional), prephenate aminotransferase, and arogenate dehydratase. In the wild type the operation of the overflow pathway is restrained by factors that restrict early-pathway flux. These factors include the highly potent feedback control of DAHP synthase isozymes by end products as well as the strikingly variable abilities of different carbon source nutrients to supply the aromatic pathway with beginning substrates. Even in the wild type, where all allosteric regulation in intact, some phenylalanine overflow was found on glucose-based medium, but not on fructose-based medium. This carbon source-dependent difference was much more exaggerated in each class of regulatory mutants.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1983

Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus.

Alcaligenes eutrophus formed ribulosebisphosphate carboxylase (RuBPCase; EC 4.1.1.39) when grown on fructose. Addition of sodium fluoride (NaF) to fructose minimal medium resulted in a slightly decreased growth rate and a rapid fivefold increase in RuBPCase specific activity. With citrate, a glucogenic carbon source, RuBPCase was also formed, However, addition of NaF to cells growing on citrate resulted in a 50% decrease in RuBPCase specific activity. Among the enzymes of fructose catabolism, NaF (10 mM) inhibited enolase in vitro by 98% and gluconate 6-phosphate dehydratase by 87%. Inhibition of the dehydratase by NaF was insignificant in vivo, as determined with a mutant defective in phosphoglycerate mutase activity. Growth of this mutant on fructose was not inhibited by NaF, and only a minor increase in RuBPCase activity was observed. From these results, we concluded that the product of the enolase reaction, phosphoenolpyruvate, played a role in RuBPCase formation. Addition of H2 or formate to the wild type growing on fructose or citrate did not affect the growth rate but resulted in rapid formation of RuBPCase activity. Mutants impaired in H2 metabolism formed RuBPCase at a low rate during growth on fructose plus H2 but at a high rate on formate. Apparently, additional reductant from H2 or formate metabolism induced RuBPCase formation in A. eutrophus.

Alcaligenes↗

PubMed1989

The Bacillus subtilis spo0B stage 0 sporulation operon encodes an essential GTP-binding protein.

Transcription of the spo0B gene and genes downstream of it was investigated by S1 nuclease protection experiments. The spo0B gene was transcribed from a single promoter, and this transcript extended through a gene, obg, coding for a 47,668 Mr protein. Transcription of this operon ended in a stem-loop structure. The sequence of the deduced obg protein contained a region with homology to known GTP-binding proteins in the nucleotide-binding regions. The amino-terminal portion of this protein showed homology to mammalian collagen, suggesting a structural role. The purified obg protein was shown to bind [alpha-32P]GTP in vitro. Several attempts to inactivate the obg gene were unsuccessful, indicating that the obg gene product was essential for growth. The possible function of this protein and its relationship to RAS-like proteins and sporulation was discussed. Immediately downstream of the obg gene were two genes involved in phenylalanine biosynthesis, pheB and pheA. The pheA gene coded for monofunctional prephenate dehydratase, on the basis of the high homology of the deduced amino acid sequence to prephenate dehydratases of bacterial origin. The sequence of the pheB gene product was not homologous to chorismate mutase, and its function remains unknown. Transcription of the phe genes was shown to begin at the stem-loop structure between obg and pheB. The possibility was entertained that phe gene transcription arises from processing or antitermination of the spo0B transcript.

Amino Acid Sequence↗

PubMed1990

Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.

Blotting, Southern↗

PubMed1992

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.

Amino Acid Sequence↗

PubMed1994

Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis.

The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.

Acetyltransferases↗

PubMed1994

Pathway of glycogen metabolism in Methanococcus maripaludis.

Methanococcus maripaludis, a facultatively autotrophic archaebacterium that grows with H2 or formate as the electron donor, does not assimilate sugars and other complex organic substrates. However, glycogen is biosynthesized intracellularly and commonly reaches values of 0.34% of the cellular dry weight in the early stationary phase. To determine the pathway of glycogen catabolism, specific enzymes of sugar metabolism were assayed in cell extracts. The following enzymes were found (specific activity in milliunits per milligram of protein): glycogen phosphorylase, 4.4; phosphoglucomutase, 10; glucose-6-phosphate isomerase, 9; 6-phosphofructokinase, 5.6, fructose-1,6-bisphosphatase, 10; fructose-1,6-bisphosphate aldolase, 4.2; triosephosphate isomerase, 44; glyceraldehyde-3-phosphate dehydrogenase, 26; phosphoglycerate kinase, 20; phosphoglycerate mutase, 78; enolase, 107; and pyruvate kinase, 4.0. Glyceraldehyde-3-phosphate dehydrogenase was NADP+ dependent, and the pyruvate kinase required MnCl2. The 6-phosphofructokinase had an unusually low pH optimum of 6.0. Four nonoxidative pentose-biosynthetic enzymes were found (specific activity in milliunits per milligram of protein): transketolase, 12; transaldolase, 24; ribulose-5-phosphate-3-epimerase, 55; and ribulose-5-phosphate isomerase, 100. However, the key enzymes of the oxidative pentose phosphate pathway, the reductive pentose phosphate pathway, and the classical and modified Entner-Duodoroff pathways were not detected. Thus, glycogen appears to be catabolized by the Embden-Meyerhoff-Parnas pathway. This result is in striking contrast to the nonmethanogenic archaebacteria that have been examined, among which the Entner-Doudoroff pathway is common. A dithiothreitol-specific NADP(+)-reducing activity was also found (8.5 mU/mg of protein). Other thiol compounds, such as cysteine hydrochloride, reduced glutathione, and 2-mercaptoethanesulfonic acid, did not replace dithiothreitol for this activity. The physiological significance of this activity is not known.

Dithiothreitol↗

PubMed1994

The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation.

Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination. In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation. By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell. Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid. The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium. We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.

Bacillus megaterium↗

PubMed2000

Metabolic pathways for cytotoxic end product formation from glutamate- and aspartate-containing peptides by Porphyromonas gingivalis.

Metabolic pathways involved in the formation of cytotoxic end products by Porphyromonas gingivalis were studied. The washed cells of P. gingivalis ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of P. gingivalis. P. gingivalis cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.

Acetic Acid↗

PubMed2000

Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12.

Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in Escherichia coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes nbzC, nbzD, and nbzE, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb SnaBI-SmaI fragment of pNB1 and clustered in the order nbzC-nbzD-nbzE as an operon. The nbzCDE genes, which are transcribed in the opposite direction of the nbzA gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.

Cloning, Molecular↗

PubMed2003

Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis.

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg(2+) ion.

Amino Acid Sequence↗

PubMed2003

Genetic loci for coaggregation receptor polysaccharide biosynthesis in Streptococcus gordonii 38.

The cell wall polysaccharide of Streptococcus gordonii 38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAcbeta1-->3Gal-containing recognition motif. The same RPS has now been identified from S. gordonii AT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize the S. gordonii 38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designated galE1, was identified as a pseudogene in the galactose operon, and the second, designated galE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e., rmlA, rmlC, and rmlB). Insertional inactivation of galE2 abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of the galE1 pseudogene in this galE2 mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production by S. gordonii 38 depends on the dual specificity of the epimerase encoded by galE2.

Bacterial Adhesion↗

PubMed2004

Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.

Amino Acids, Aromatic↗

PubMed1966

Control of aromatic acid biosynthesis in Bacillus subtilis: sequenial feedback inhibition.

Nester, E. W. (University of Washington, Seattle), and R. A. Jensen. Control of aromatic acid biosynthesis in Bacillus subtilis: sequential feedback inhibition. J. Bacteriol. 91:1594-1598. 1966.-The three major end products of aromatic acid synthesis, tyrosine, phenylalanine, and tryptophan, were tested for their ability to inhibit the first enzymes of the three terminal branches of the pathway as well as the enzyme common to both tyrosine and phenylalanine synthesis. Tyrosine inhibits the activity of prephenate dehydrogenase and also prephenate dehydratase to a limited extent. Phenylalanine inhibits the activity of prephenate dehydratase and, at much higher concentrations, prephenate dehydrogenase. Tryptophan inhibits the activity of anthranilate synthetase and, to some extent, prephenate dehydrogenase and prephenate dehydratase. Chorismate mutase is not inhibited by either 1 mm tyrosine or 1 mm phenylalanine when these are present singly or together in the reaction mixture. The significance of the feedback control of the terminal branches to the feedback control of that part of the pathway common to the synthesis of all three amino acids is discussed.

Bacillus subtilis↗

PubMed1969

Divergent metabolic pathways for propane and propionate utilization by a soil isolate.

The metabolism of propane and propionate by a soil isolate (Brevibacterium sp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme in n-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of (14)CO(2) into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-(14)C, -2-(14)C, and -3-(14)C as substrate. Propane -2-(14)C was shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO(2) would yield the inducer for the isocitrate lyase present in propane-grown cells.

Alkanes↗

PubMed1995

The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein.

The GCR1 gene product is required for maximal transcription of yeast glycolytic genes and for growth of yeast strains in media containing glucose as a carbon source. Dominant mutations in two genes, SGC1 and SGC2, as well as recessive mutations in the SGC5 gene were identified as suppressors of the growth and transcriptional defects caused by a gcr1 null mutation. The wild-type and mutant alleles of SGC1 were cloned and sequenced. The predicted amino acid sequence of the SGC1 gene product includes a region with substantial similarity to the basic-helix-loop-helix domain of the Myc family of DNA-binding proteins. The SGC1-1 dominant mutant allele contained a substitution of glutamine for a highly conserved glutamic acid residue within the putative basic DNA binding domain. A second dominant mutant, SGC1-2, contained a valine-for-isoleucine substitution within the putative loop region. The SGC1-1 dominant mutant suppressed the GCR1 requirement for enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase gene expression. Expression of the yeast enolase genes was reduced three- to fivefold in strains carrying an sgc1 null mutation, demonstrating that SGC1 is required for maximal enolase gene expression. Expression of the enolase genes in strains carrying gcr1 and sgc1 double null mutations was substantially less than observed for strains carrying either null mutation alone, suggesting that GCR1 and SGC1 function on parallel pathways to activate yeast glycolytic gene expression.

Alleles↗

PubMed2002

Identification, cDNA cloning, and targeted deletion of p70, a novel, ubiquitously expressed SH3 domain-containing protein.

In a screen for proteins that interact with Jak2, we identified a previously uncharacterized 70-kDa protein and cloned the corresponding cDNA. The predicated sequence indicates that p70 contains an SH3 domain and a C-terminal domain with similarities to the catalytic motif of phosphoglycerate mutase. p70 transcripts were found in all tissues examined. Similarly, when an antibody raised against a C-terminal peptide to analyze p70 protein expression was used, all murine tissues examined were found to express p70. To investigate the in vivo role of p70, we generated a p70-deficient mouse strain. Mice lacking p70 are viable, develop normally, and do not display any obvious abnormalities. No differences were detected in various hematological parameters, including bone marrow colony-forming ability, in response to cytokines that utilize Jak2. In addition, no impairment in B- and T-cell development and proliferative ability was detected.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Receptor-mediated endocytosis of cobalamin (vitamin B12).

Dietary cobalamin (Cbl) (vitamin B12) is utilized as methyl-Cbl and the coenzyme 5'-deoxyadenosyl Cbl by cells of the body that have the enzymes methionine synthase and methyl malonyl CoA mutase, which convert homocysteine to methionine and methyl malonyl CoA to succinyl CoA, respectively. Prior to conversions and utilizations as the active alkyl forms of Cbl, dietary Cbl is absorbed and transported across cellular plasma membranes by two receptor-mediated events. First, dietary and biliary Cbl bound to gastric intrinsic factor (IF) presented apically to the ileal absorptive enterocytes is transported to the circulation by receptor-mediated endocytosis via apically expressed IF-Cbl receptor. Second, Cbl bound to plasma transcobalamin (TC) II is taken up from the circulation by all cells via a TC II receptor expressed in the plasma membrane of these cells, and in polarized cells via a TC II receptor expressed in the basolateral membranes. This review updates recent work and focuses on (a) the molecular and cellular aspects of Cbl binding protein ligands, IF and TC II, and their cell-surface receptors, IF-Cbl receptor and TC II receptor; (b) the cellular sorting pathways of internalized Cbl bound to IF and TC II in polarized epithelial cells; and (c) the absorption and transport disorders that cause Cbl deficiency.

Amino Acid Sequence↗