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PubMed1977

Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase.

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1994

A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.

2,3-Diphosphoglycerate↗

PubMed1997

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions.

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Chorismate Mutase↗

PubMed2004

Cofactor-independent phosphoglycerate mutase has an essential role in Caenorhabditis elegans and is conserved in parasitic nematodes.

Phosphoglycerate mutases catalyze the interconversion of 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. They exist in two unrelated forms that are either cofactor (2,3-diphosphoglycerate)-dependent or cofactor-independent. The two enzymes have no similarity in amino acid sequence, tertiary structure, or catalytic mechanism. Certain organisms including vertebrates have only the cofactor-dependent form, whereas other organisms can possess the independent form or both. Caenorhabditis elegans has been predicted to have only independent phosphoglycerate mutase. In this study, we have cloned and produced recombinant, independent phosphoglycerate mutases from C. elegans and the human-parasitic nematode Brugia malayi. They are 70% identical to each other and related to known bacterial, fungal, and protozoan enzymes. The nematode enzymes possess the catalytic serine, and other key amino acids proposed for catalysis and recombinant enzymes showed typical phosphoglycerate mutase activities in both the glycolytic and gluconeogenic directions. The gene is essential in C. elegans, because the reduction of its activity by RNA interference led to embryonic lethality, larval lethality, and abnormal body morphology. Promoter reporter analysis indicated widespread expression in larval and adult C. elegans with the highest levels apparent in the nerve ring, intestine, and body wall muscles. The enzyme was found in a diverse group of nematodes representing the major clades, indicating that it is conserved throughout this phylum. Our results demonstrate that nematodes, unlike vertebrates, utilize independent phosphoglycerate mutase in glycolytic and gluconeogenic pathways and that the enzyme is probably essential for all nematodes.

Amino Acid Sequence↗

PubMed2005

Nitric oxide inhibits mammalian methylmalonyl-CoA mutase.

Methylmalonyl-CoA mutase is a key enzyme in intermediary metabolism, and children deficient in enzyme activity have severe metabolic acidosis. We found that nitric oxide (NO) inhibits methylmalonyl-CoA mutase activity in rodent cell extracts. The inhibition of enzyme activity occurred within minutes and was not prevented by thiols, suggesting that enzyme inhibition was not occurring via NO reaction with cysteine residues to form nitrosothiol groups. Enzyme inhibition was dependent on the presence of substrate, implying that NO was reacting with cobalamin(II) (Cbl(II)) and/or the deoxyadenosyl radical (.CH(2)-Ado), both of which are generated from the co-factor of the enzyme, 5'-deoxyadenosyl-cobalamin (AdoCbl), on substrate binding. Consistent with this hypothesis was the finding that high micromolar concentrations (> or =600 microm) of oxygen also inhibited enzyme activity. To study the mechanism of NO reaction with AdoCbl, we simulated the enzymatic reaction by photolyzing AdoCbl, and found that even at low NO concentrations, NO reacted with both the generated Cbl(II) and .CH(2)-Ado indicating that NO could effectively compete with the back formation of AdoCbl. Thus, NO inhibition of methylmalonyl-CoA mutase appeared to be from the reaction of NO with both AdoCbl intermediates (Cbl(II) and .CH(2)-Ado) generated during the enzymatic reaction. The inhibition of methylmalonyl-CoA mutase by NO was likely of physiological relevance because a NO donor inhibited enzyme activity in intact cells, and scavenging NO from cells or inhibiting cellular NO synthesis increased methylmalonyl-CoA mutase activity when measured subsequently in cell extracts.

Animals↗

PubMed1982

The amino acid sequence of yeast phosphoglycerate mutase.

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8-12 residue peptide from the C-terminus.

Amino Acid Sequence↗

PubMed1996

Adenosylcobalamin-dependent methylmalonyl-CoA mutase isozymes in the photosynthetic protozoon Euglena gracilis Z.

The photosynthetic protozoon Euglena gracilis Z contains adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) involved in propionate metabolism. The specific activity of the Euglena mutase was about 6.5-fold greater in propionate-adapted Euglena cells than in photoautotrophic cells (control). Although the control cells contained only one mutase (apparent M(r) 72,000), the propionate-adapted cells contained two mutases with M(r) values of 72,000 and 17,000; both enzymes were located in the mitochondria. These results provide evidence that propionate-adapted Euglena contains two MCM isozymes. The induced mutase (M(r) 17,000) permits photoassimilation of propionate.

Animals↗

PubMed1978

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.

Acyl Coenzyme A↗

PubMed1989

Purification, characterization and immunological properties of 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize (Zea mays) seeds.

2,3-Bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) was purified and characterized from maize. SDS electrophoresis showed only one band with a molecular mass of 64 kDa, similar to that determined for the native enzyme by gel-filtration chromatography. The kinetic constants were similar to those reported for wheat germ phosphoglycerate mutase. Rabbit antiserum against maize phosphoglycerate mutase possesses a high degree of specificity. It also reacts with the wheat germ enzyme but fails to react with other cofactor-independent or cofactor-dependent phosphoglycerate mutases. Cell-free synthesis experiments indicate that phosphoglycerate mutase from maize is not post-translationally modified.

2,3-Diphosphoglycerate↗

PubMed1993

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.

2,3-Diphosphoglycerate↗

PubMed1976

Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes.

Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given.

Amino Acids↗

PubMed2003

Reconstitution of UDP-galactopyranose mutase with 1-deaza-FAD and 5-deaza-FAD: analysis and mechanistic implications.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by a FAD-dependent UDP-Galp mutase. When the enzyme is reduced by sodium dithionite, its catalytic efficiency increases significantly. Since the overall transformation exhibits no net change in the redox state of the parties involved, how the enzyme-bound FAD plays an active role in the reaction mechanism is puzzling. In this paper, we report our study of the catalytic properties of UDP-Galp mutase reconstituted with deaza-FADs. It was found that the mutase reconstituted with FAD or 1-deazaFAD has comparable activity, while that reconstituted with 5-deazaFAD is catalytically inactive. Because 5-deazaFAD is restricted to net two-electron process, yet FAD and 1-deazaFAD can undergo concerted two-electron as well as stepwise one-electron redox reactions, the above results support a radical mechanism for the mutase catalyzed reaction. In addition, the activity of the mutase reconstituted with FAD was found to increase considerably at high pHs. These observations have allowed us to propose a new mechanism involving one-electron transfer from the reduced FAD to an oxocarbenium intermediate generated by C-1 elimination of UDP to give a hexose radical and a flavin semiquinone. Subsequent radical recombination leads to a coenzyme-substrate adduct which may play a central role to facilitate the opening and recyclization of the galactose ring. A deprotonation step, accompanied or followed the electron transfer step, to increase the nucleophilicity of the flavin radical anion may account for the activity enhancement at pH > 8.

Coenzymes↗

PubMed1999

Helix swapping between two alpha/beta barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg(2+)-oxalate.

BACKGROUND: Phosphonate compounds are important secondary metabolites in nature and, when linked to macromolecules in eukaryotes, they might play a role in cell signaling. The first obligatory step in the biosynthesis of phosphonates is the formation of a carbon-phosphorus bond by converting phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr), a reaction that is catalyzed by PEP mutase. The PEP mutase functions as a tetramer and requires magnesium ions (Mg2+). RESULTS: The crystal structure of PEP mutase from the mollusk Mytilus edulis, bound to the inhibitor Mg(2+)-oxalate, has been determined using multiwavelength anomalous diffraction, exploiting the selenium absorption edge of a selenomethionine-containing protein. The structure has been refined at 1.8 A resolution. PEP mutase adopts a modified alpha/beta barrel fold, in which the eighth alpha helix projects away from the alpha/beta barrel instead of packing against the beta sheet. A tightly associated dimer is formed, such that the two eighth helices are swapped, each packing against the beta sheet of the neighboring molecule. A dimer of dimers further associates into a tetramer. Mg(2+)-oxalate is buried close to the center of the barrel, at the C-terminal ends of the beta strands. CONCLUSIONS: The tetramer observed in the crystal is likely to be physiologically relevant. Because the Mg(2+)-oxalate is inaccessible to solvent, substrate binding and dissociation might be accompanied by conformational changes. A mechanism involving a phosphoenzyme intermediate is proposed, with Asp58 acting as the nucleophilic entity that accepts and delivers the phosphoryl group. The active-site architecture and the chemistry performed by PEP mutase are different from other alpha/beta-barrel proteins that bind pyruvate or PEP, thus the enzyme might represent a new family of alpha/beta-barrel proteins.

Animals↗

PubMed2001

Mechanistic investigation of UDP-galactopyranose mutase from Escherichia coli using 2- and 3-fluorinated UDP-galactofuranose as probes.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.

Bacterial Proteins↗

PubMed2003

Properties of phosphoenolpyruvate mutase, the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi.

Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.

Amino Acid Sequence↗

PubMed2006

A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose.

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.

Amino Acid Sequence↗

PubMed1978

Comparison of chorismate mutase isozyme patterns in selected plants.

A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sativa L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyllophyta (a Selaginella) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase.

Journal Article↗

PubMed1988

Increased Chorismate Mutase Levels as a Response to Wounding in Solanum tuberosum L. Tubers.

Discs excised from Solanum tuberosum L. cv White Rose tubers demonstrated a 4.5-fold increase in chorismate mutase activity 48 hours after excision. Incubation in the presence of cycloheximide (25 micromolar) or actinomycin D (100 micromolar) completely inhibited the wound response suggesting de novo synthesis of chorismate mutase. Ratios of activity in the presence of the activator tryptophan to that in the absence of tryptophan remained relatively constant during the induction period. This indicated either a constant ratio of tryptophan sensitive to tryptophan insensitive isozymes, or that only one form of chorismate mutase was present. Chromatography of crude extracts on three different columns yielded only one peak of chorismate mutase activity, activated by tryptophan in each case. Incubation under white light had no effect on chorismate mutase activity when compared to dark controls.

Journal Article↗