Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids

A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids.     

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.     

Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.     

Studies on adrenal delta-aminolevulinic acid synthetase.

The presence of δ-aminolevulinic acid synthetase (EC 2.3.1.37) in rat and bovine adrenals has been demonstrated. When untreated animals are employed, the activity of δ-aminolevulinic acid synthetase in rat and bovine adrenal homogenates is comparable to the activity found in hepatic homogenates. Adrenal δ-aminolevulinic acid synthetase is localized in the mitochondrial fraction and appears to be refractory to induction by agents that induce the hepatic enzyme. Starvation of rats increased adrenal δ-aminolevulinic acid synthetase activity without altering the activity of the hepatic enzyme. Treatment of rats with adrenocorticotropin also dramatically increased adrenal δ-aminolevulinic acid synthetase activity. These results suggest that the adrenal enzyme may be controlled by factors that differ from those which regulate the activity of the hepatic enzyme.     

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Effects of fatty acids on activity and calmodulin binding of Ca2+-ATPase of human erythrocyte membranes

Activation and inhibition of Ca2+-ATPase of calmodulin-depleted human erythrocyte membranes by oleic acid and a variety of other fatty acids have been measured. Low concentrations of oleic acid stimulate the enzyme activity, both in the presence and in the absence of calmodulin. Concomitantly, the affinity of the membrane bound enzyme to calmodulin progressively decreases due to competitive interactions of calmodulin and oleic acid with the enzyme. Removal of oleic acid from the membrane by serum albumin extinguishes the activating effect of oleic acid and restores the ability of the enzyme to bind calmodulin with high affinity. High concentrations of oleic acid induce an almost complete and irreversible loss of enzyme activity which cannot be abolished by removal of oleic acid. Despite a complete loss of enzyme activity, binding of calmodulin to membranes is approximately normal after removal of oleic acid. Activities of (Na+ + K+)-ATPase, Mg2+-ATPase and acetylcholine esterase, as well as the total protein content, show no gross changes upon treatment of membranes with increasing amounts of oleic acid, which seems to exclude that membrane solubilisation by oleic acid causes an inactivation of the enzyme.     

Purification and Properties of a Dehyrodicaffeic Acid Dilactone-forming Enzyme from a Mushroom,Inonotus sp. K-1410

A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C.     

Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Evidence that the enzyme is a homodimer of identical subunits homologous to rat liver-specific alloantigen F.

4-Hydroxyphenylpyruvic acid dioxygenase is an important enzyme in tyrosine catabolism in most organisms. From porcine and human liver cDNA libraries we isolated complementary DNA inserts for the enzyme. Protein sequence analysis of the porcine enzyme revealed a block of the amino terminus of the mature enzyme. Comparison of the amino acid sequence determined by Edman degradation of peptides derived from porcine liver 4-hydroxyphenylpyruvic acid dioxygenase with the nucleotide sequences revealed the primary structure of the porcine and human enzymes. The mature human and porcine enzymes have an 89% amino acid sequence identity in amino acid residues and are composed of 392 amino acid residues. A computer-assisted homology search revealed that the enzyme is 88% identical in amino acid sequence to rat liver-specific alloantigen F. A monoclonal antibody (mob 51), which can immunoprecipitate both the human and porcine enzymes, was developed. Cultured BMT-10 cells transfected with the cDNA insert of the human enzyme, using the expression vector pCAGGSneodE, produced a polypeptide with an M(r) of 43,000, which was immunoprecipitated with mob 51. Enzymic activity of the enzyme was detected in the transfected cells but not in the mock transfected cells. These findings suggest that the human 4-hydroxyphenylpyruvic acid dioxygenase is a homodimer of two identical subunits with an M(r) of 43,000. Liver-specific alloantigen F seems to be closely related to the enzyme or possibly to the subunit of the enzyme itself. Elucidation of the complete amino acid sequence of the enzyme is expected to reveal structure-function relationships of this metabolically important enzyme and to shed light on inherited disorders related to tyrosine metabolism, especially tyrosinemia types 1 and 3.     

Acetyl-coenzyme-A carboxylase in cultured hepatocytes. Effects of exogenous fatty acids on the content, synthesis and degradation of the enzyme.

Studies were made on the content, synthesis and degradation of acetyl-coenzyme-A carboxylase in JTC-25 - P3 cells, hepatocytes which can be maintained in a protein-free and lipid-free chemically defined medium. The addition of corn oil or fatty acid to the medium resulted in a decrease in the activity level of the enzyme without impairing the viability of cells. All the fatty acids tested exhibited this effect, although linoleic acid and oleic acid were more effective than palmitic acid, stearic acid and arachidonic acid. Immunochemical titration and Ouchterlony double-diffusion analysis indicated that the decrease in the activity level of the enzyme observed in cells incubated in medium supplemented with fatty acid can be ascribed to a reduction of the quantity of the enzyme. Isotopic leucine incorporation studies with the use of immunochemical techniques demonstrated that this reduction of the enzyme content is due to a decrease in the rate of synthesis of the enzyme. The rate of degradation of the enzyme was essentially unaffected, the half-life being 25 and 28 h, respectively, in cells incubated in the presence and absence of fatty acid. It was shown that most of the isotopic fatty acid added to the medium was incorporated into cellular phospholipids, while a very small portion of it was recovered in triglyceride and nonesterified fatty acid.     

Green butyryl-coenzyme A dehydrogenase. An enzyme–acyl-coenzyme A complex

1. Butyryl-CoA dehydrogenase from Peptostreptococcus elsdenii forms very tightly bound complexes with various acyl-CoA compounds. Spectra in some cases merely show resolution of the 450nm band, but those with acetoacetyl-, pent-2-enoyl- and 4-methylpent-2-enoyl-CoA show long-wavelength bands similar to the 710nm band of native enzyme. These complexes are formed instantaneously by the yellow form of the enzyme and much more slowly by the green form. 2. An acid extract of the green enzyme reconverts the yellow into the green form. 3. Hydroxylamine makes irreversible the otherwise reversible conversion of the green enzyme into the yellow form by phenylmercuric acetate. 4. Amino acid analysis for taurine and beta-alanine shows approx. 1mol of CoA/mol of flavin in green enzyme. Anaerobic dialysis of reduced enzyme removes the CoA. On acid precipitation of green enzyme the CoA is found only in the supernatant. 5. It is concluded that native green enzyme is probably complexed with unsaturated acyl-CoA. This is shown to be consistent with findings of other workers. Catalytic activity requires displacement of the acyl-CoA, which is therefore likely to be a potent inhibitor. 6. An explanation is offered for the irreversible conversion of green into yellow enzyme by sodium dithionite. 7. The enzyme displays a feeble, previously undetected, activity towards beta-hydroxybutyryl-CoA. 8. The product of oxidation of pent-4-enoyl-CoA forms a complex with reduced enzyme and strongly inhibits reoxidation of the FAD. This may contribute to inhibition of fatty acid oxidation by pent-4-enoic acid in mammals.     

Molecular and catalytic properties of a butyrylesterase from human red cells and brain

A butyrylesterase from human red cells was prepared to homogeneity using DEAE-cellulose, Ultrogel ACA-34, DEAE-Sephacel, and precipitation with 1.5 M (NH4)2SO4. The yield was 25-35% relative to the enzyme activity of the hemolysate. Because of its preference for butyric acid esters the enzyme was designated a butyrylesterase. With alpha-naphthyl butyrate the Km was 7.6 microM and the kcat, 48 s-1. The molecular weight was 340,000 and the subunit weight 85,000, indicating a tetrameric structure. The isoelectric pH was 4.0. The enzyme preparation did not contain cystine. Sialic acid or other carbohydrate components could not be detected. The enzyme was irreversibly inhibited by organophosphate esters and the second-order rate constant was 192 M-1 s-1 for diethyl p-nitrophenyl phosphate. For the brain enzyme the constant was 206 M-1 s-1. The enzyme was irreversibly inhibited by sulfhydryl reagents, indicating that the enzyme is a sulfhydryl-dependent serine esterase. The enzyme was identical to the butyrylesterase from human brain, and the two enzymes were immunochemically identical. An amino acid ester has been shown to be split at a higher rate than butyric acid esters; however, the specificity constant (kcat/Km) was lower for the amino acid ester than for the butyric acid ester. The enzyme did not exhibit amidase activity.     

Isolation and amino acid sequence of a peptide containing an epoxide-reactive residue from the thermolysin-digest of Scytalidium lignicolum acid protease B

Scytalidium lignicolum acid protease B, a pepstatin-insensitive acid protease, was modified by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) with the concomitant loss of its enzyme activity, and an EPNP-labeled peptide was isolated from the thermolysin-digest of the modified enzyme by HPLC. The amino acid sequence of the peptide was determined to be Ile-Leu-Glu-Thr-Gly, which corresponds to the sequence of residue Nos. 51-55 of the enzyme. The results of treatment of the labeled peptide with hydroxylamine suggested that the EPNP moiety is ester-linked to Glu53 of the enzyme. The amino acid sequence around Glu53 of the acid protease B showed high homology with those around the active site Asp residues of calf chymosin and porcine pepsin. These results show that it is highly possible that Glu53 of the acid protease B is one of the amino acid residues involved in its catalytic activity.     

Biosynthesis of polymyxin E by a cell-free enzyme system. IV. Acylation of enzyme-bound L-2,4-diaminobutyric acid

An enzyme fraction, which catalyzes the ATP-PPi exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640,000 by Sepharose 4B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2,4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.     

Oxodiene formation during the Vicia sativa lipoxygenase-catalyzed reaction: occurrence of dioxygenase and fatty acid lyase activities associated in a single protein

An enzyme with at least dual activities, lipoxygenase and fatty acid lyase, has been isolated from Vicia sativa seeds. The enzyme utilizes directly linoleic acid as substrate. The enzyme had a pH optimum at 5.8 for the two activities and converted linoleic acid into two products: 9-hydroperoxylinoleic acid and trans-2, cis-4 decadienal. The enzyme does not act on 13- or 9- fatty acid hydroperoxide isomers. An enzymatic reaction for the biogenesis of trans-2, cis-4- decadienal is proposed. This involves the synthesis of an intermediate peroxyl radical due to oxygen insertion in carbon 9 of linoleic acid. This intermediate peroxyl radical may be converted into 9-HPOD and 2,4-decadienal.     

Solubilization and characterization of a cell wall-bound trehalase from ascospores of the fission yeast Schizosaccharomyces pombe

The genome of the fission yeast Schizosaccharomyces pombe lacks sequence homologs to ath1 genes coding for acid trehalases in other yeasts or filamentous fungi. However, acid trehalase activity is present at the spore stage in the life cycle of the fission yeast. The enzyme responsible for this activity behaves as a surface enzyme covalently linked to the spore cell walls in both wild-type and ntp1 mutant strains devoid of neutral trehalase. Lytic treatment of particulated cell wall fractions allowed the solubilization of the enzyme into an active form. We have characterized this soluble enzyme and found that its kinetic parameters, optimum pH and temperature, thermal denaturation and salt responses are closely similar to other conventional acid trehalases. Hence, this rather unusual enzyme can be recognized as acid trehalase by its biochemical properties although it does not share genetic homology with other known acid trehalases. The potential role of such acid trehalase in the mobilization of trehalose is discussed.     

Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.     

Truncated forms of the recombinant Escherichia coli ADP-glucose pyrophosphorylase: the importance of the N-terminal region for allosteric activation and inhibition

Truncated forms of Escherichia coli ADPglucose pyrophosphorylase were constructed using recombinant DNA techniques. A truncated form of the enzyme having the first 11 amino acid residues from the N-terminus and 2 amino acid residues from the C-terminus deleted was found to be highly active in absence of activator. A 1.6-fold activation by 1.5 mM fructose 1,6 bis-phosphate was observed for the truncated enzyme as compared to the 30-fold activation seen for the intact enzyme. Inhibition of the truncated enzyme by AMP was less than that seen with the intact enzyme. Similar properties were displayed by an enzyme truncated only at the N-terminal. Conversely, the C-terminal truncated enzyme shortened by 2 amino acid residues at the C-terminus is as sensitive as the intact enzyme to activation and inhibition. These results suggest that the N-terminal region is required for allosteric regulation of the enzyme.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzymatic oxidation of kojic acid to comenic aldehyde was found in the decomposition process of kojic acid by Arthrobacter ureafaciens strain (K-l), a kojic acid decomposing bacteria.

This enzyme was (probable a new type of non-heme iron protein) is assumed to catalyze the dehydrogenation of kojic acid, while the ferric ion contained in the enzyme is considered to serve as an acceptor of hydrogen released from kojic acid. The resulted ferrous ions are oxidized either by molecular oxygen under aerobic conditions or by NAD under anaerobic conditions, accompanying hydrogen peroxide in the former and reduced NAD in the latter. The enzyme was partially purified by using ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and column chromatography with DEAE-Sephadex A-50. The activity increased to 85 fold, compared with crude extracts and the recovery of the activity was 33.9%. The optimum pH of the reaction was 7.75. The enzyme was inactivated by PCMB, and unstable upon heat treatment. A loss of about 50% of the activity was caused by heating at 35%C for 5 min, but some reducing agents protected the enzyme from PCMB inhibition and the heat inactivation. Not only kojic acid, but also benzyl kojic acid or 5-methoxy kojic acid may be substrates. Km value for kojic acid was 1.43 × 10^?5m. The molecular weight of the enzyme was estimated to be about 55,000 and the enzyme contained about two atoms of iron in one molecule. The reaction mechanism for kojic acid oxidase is discussed.     

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.