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.     

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.     

Optimization of a process for the production of (R)-2-hydroxy-4-phenylbutyric acid--an intermediate for inhibitors of angiotensin converting enzyme.

(R)-2-Hydroxy-4-phenylbutyric acid, an intermediate in the manufacture of inhibitors of angiotensin converting enzyme, can be produced continuously in an enzyme membrane reactor by enzymatic reduction of its corresponding alpha-keto acid. D-Lactate dehydrogenase (D-LDH) from Staphylococcus epidermidis was chosen as the most appropriate enzyme to carry out the NADH-dependent reduction. Formate dehydrogenase (FDH) was used for NADH regeneration. Detailed kinetic measurements and a mathematical model for the coupled enzyme reactions were applied to calculate the optimal conditions for continuous production of the alpha-hydroxy acid. A mass of 1 kg [corrected] (R)-2-hydroxy-4-phenylbutyric acid was synthesized in a 220 ml enzyme membrane reactor over a period of 4 weeks. A mean space-time-yield of 165 g l-1 d-1 was achieved at low enzyme consumptions of 150 U kg-1 alpha-hydroxy acid for FDH and D-LDH.     

Leukotriene A4 hydrolase in the human lung. Inactivation of the enzyme with leukotriene A4 isomers

Leukotriene A4 hydrolase from the human lung was purified to apparent homogeneity. The molecular weight (68,000-71,000), the amino acid composition, and the N-terminal amino acid sequence were similar to those of the human neutrophil enzyme but different from those of human erythrocyte enzyme. The lung enzyme was inactivated by its substrate, leukotriene A4. To elucidate the substrate and the inactivator specificity of this enzyme, we synthesized various geometric and positional isomers of leukotriene A4. 14,15-Leukotriene A4, leukotriene A4 methyl ester, and geometric isomers of leukotriene A4 could not serve as substrates, but they inactivated the enzyme. On the other hand, styrene oxide and (5S)-trans-5,6-oxide-8,10,14-cis-12-trans-eicosatetraenoic acid neither served as substrates nor inactivated the enzyme. These results indicate that whereas allylic epoxide structures of arachidonic acids are responsible for inactivation of the enzyme, the free carboxylic acid, 5,6-oxide, and the tetraene structure with the 7,9-trans-11,14-cis configuration are required as a substrate for leukotriene A4 hydrolase.     

Human glucose 6-phosphate dehydrogenase: Purification and characterization of negro type variant (A+) and comparison with normal enzyme (B+)

Human glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.49) (A+), an electrophoretically distinguishable variant found in Negroes, was purified by column chromatographic techniques. The sedimentation patterns of analytical ultracentrifugation and interference patterns of sedimentation equilibrium indicate a homogeneous preparation. The molecular weight (by sedimentation equilibrium method) was estimated as 230,000, which was closely similar to that of the normal wild type enzyme (B+). The sedimentation constant of the variant enzyme (S _20,w=9.0) was smaller than that of the B+ enzyme (S _20,w=10.0). The molecular weight was about 45,000 in 4 mguanidine hydrochloride, indicating that the A+ enzyme, as well as the B+ enzyme, consisted of six subunits of similar size. The optimal pH of the variant enzyme was slightly higher than that of the B+ enzyme. In contrast to the B+ enzyme, magnesium ion increased the A+ enzyme activity with NAD as substrate. The Michaelis constants and the turnover rate were similar to those of the B+ enzyme. The A+ enzyme was serologically indistinguishable from the B+ enzyme when the anti-B+ serum was used as antibody. No significant difference was found in the amino acid composition of acid hydrolysates of the B+ and the A+ enzymes. This does not exclude an amino acid substitution, and, in fact, a single amino acid substitution, i.e., asparagine in B+ and aspartic acid in A+ enzyme, has been found and is being being reported separately.Supported by Research Grant HD-02497-01 and H-3901 from the National Institutes of Health.     

A unique enzyme from Saccharothrix sp. catalyzing d-amino acid transfer

A newly isolated actinomycete belonging to Saccharothrix sp. was found to produce a unique enzyme catalyzing d-amino acid transfer. The enzyme, which was tentatively named d-amino acid transferase, was purified 2600-fold to electrophoretic homogeneity and the molecular mass was 41 kDa. The enzyme was d-configuration specific and recognized aromatic d-amino acid esters to form oligo d-amino acid esters. d-Phenylalanine ester was favored as substrate over other d-amino acid esters. The optimum conditions for oligo d-phenylalanine ester formation by d-amino acid transferase were pH 7.0 and 40°C. The enzyme was inhibited by DAN, EPNP and DFP.     

The inhibition of staphylococcal beta-lactamase by clavulanic acid.

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.     

Identification in the mold Hypocrea jecorina of the first fungal D-galacturonic acid reductase

A d-galacturonic acid reductase and the corresponding gene were identified from the mold Hypocrea jecorina (Trichoderma reesei). We hypothesize that the enzyme is part of a fungal d-galacturonic acid catabolic pathway which has not been described previously and which is distinctly different from the bacterial pathway. H. jecorina grown on d-galacturonic acid exhibits an NADPH-dependent d-galacturonic acid reductase activity. This activity is absent when the mold is grown on other carbon sources. The d-galacturonic acid reductase was purified, and tryptic digests of the purified protein were sequenced. The open reading frame of the corresponding gene was then cloned from a cDNA library. The open reading frame was functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was purified, and the enzyme kinetics were characterized. The enzyme converts in a reversible reaction from d-galacturonic acid and NADPH to l-galactonic acid and NADP. The enzyme also exhibits activity with d-glucuronic acid and dl-glyceraldehyde.     

Purification and properties of a glucoamylase fraction from the culture filtrate of Rhizopus nodosus

A glucoamylase was isolated from the culture filtrate of Rhizopus nodosus and was separated from the acid lipase by DEAE-cellulose chromatography at pH 8.0 It was purified by Concanavalin A Sepharose 4B affinity chromatography followed by CM-Sephadex chromatography 387 fold with 30.7% yield. The homogeneity of the enzyme were confirmed by polyacrylamide gel electrophoresis and immunological studies. The different physico-chemical properties of the enzyme were studied. The molecular weight of the enzyme was found to be 71,000. Ethylenediaminetetraacetic acid had no effect on the enzyme whereas Hg2+ partially inhibited the enzyme activity. Tryptophan residues were found to be essential for the enzyme activity.     

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.     

The pectinolytic enzyme of Selenomonas ruminantium

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40°. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nona-galacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-aP-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.     

五步蛇蛇毒磷脂酶A_2的纯化及部分性质

经Sephadex G-75和QAE-Sephadex A-50离子交换层析等方法,从湖南产五步蛇(Agkistrodon acutus)蛇毒中纯化一种均一的酸性磷脂酶A_2。SDS-PAGE测得分子量为15.8kD,按氨基酸残基计算其分子量为14.352kD,IEF-PAGE测得等电点为5.32。氨基酸组份分析表明磷脂酶A_2分子由128个氨基酸残基组成,富含Asp和Glu,不含中性糖。PLA_2酶活性的最适温度为45℃,最适pH为8.5左右,没有抗胰蛋白酶的活性,具一定的热稳定性。K~+、Ca~(++)和Na~+离子激活,而Cd~(++)、Sn~(++)、Cu~(++)、Li~+、Hg(++)、Zn~(++)、Fe~(++)和Co~(++)离子可抑制或完全丧失酶活力。手工微量顺序分析测得PLA_2分子N-末端氨基酸为Leu。此酶对小白鼠的LD_(50)至少大于10mg/kg(ip)。     

The pectinolytic enzyme of Selenomonas ruminantium

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40 degrees C. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nonagalacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-alpha-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.     

cDNA cloning of rice lipoxygenase L-2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli.

A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15 degrees C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96,657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.     

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.     

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.     

On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J)

A covalent, catalytic intermediate of cytosolic liver acetyl coenzyme A: arylamine N-acetyltransferase (EC 2.3.1.5) from rapid acetylator rabbits (III/J) was isolated and chemically characterized. The active site was further studied using two covalent inhibitors, [2-3H]iodoacetic acid and bromoacetanilide. Inhibition experiments with [2-3H]iodoacetic acid at pH 6.9 showed that the incorporation of 0.7 mol of [2-3H]iodoacetic acid/mol of N-acetyltransferase led to rapid, irreversible loss of enzyme activity. Preincubation of the enzyme with acetyl coenzyme A (acetyl-CoA) completely protected against inactivation by [2-3H]iodoacetic acid. After incubating the N-acetyltransferase with [2-3H]acetyl-CoA in the absence of an acceptor amine, an acetyl-cysteinyl-enzyme intermediate was isolated and characterized. Preincubation of N-acetyltransferase with iodoacetic acid prevented the incorporation of the [2-3H]acetyl group into the enzyme. The product analog, bromoacetanilide, caused a rapid irreversible loss of N-acetyltransferase activity. The reaction was pseudo first-order and saturated at high bromoacetanilide concentrations (KI = 0.67 mM; k3 = 1 min-1). Preincubation of the enzyme with acetyl-CoA prevented inactivation by the inhibitor. The acceptor amine 4-ethylaniline did not prevent inhibition. Incorporation of the inhibitor was directly proportional to the loss of activity showing a 1:1 stoichiometry of enzyme to inhibitor. The target amino acid was identified as cysteine by amino acid analysis of inhibitor-treated enzyme.     

Purification and Properties of a Pectin trans-Eliminase in Erwin aroideae Formed in the Presence of Nalidixic Acid

A strain of Erwinia aroideae produces a remarkable amount of pectolytic enzyme when the organism was induced by nalidixic acid for the bacteriocin production. This pectolytic enzyme was purified approximately 60-fold from the induced medium by carboxymethyl-cellulose and Sephadex G–75 gel column chromatographies after batchwise treatment with carboxymethyl- and diethylaminoethyl-celluloses. The purified enzyme was almost homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a molecular weight of about 28,000 to 32,000 was determined for this enzyme. The optimum pH of the enzyme activity was about 8.0 to 8.2. The purified enzyme produced reaction products from pectin and methoxylated pectic acid which had a strong absorption at 235 nm indicating a trans-eliminase reaction. Pectin or pectic acid with higher methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectic acid was a substrate. The limit of degradation of pectin and pectic acid with higher methoxyl content (90% esterified) by the enzyme were 6.5% and 43%, respectively. It was concluded that the enzyme is a new endo-pectin trans-eliminase from bacterial origin.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Partial purification and characterization of 7 alpha-hydroxysteroid dehydrogenase from rat liver microsomes

An NADPH-dependent 7 alpha-hydroxysteroid dehydrogenase acting on 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was partially purified 160-fold with a yield of 13% from rat liver microsomes using DEAE-cellulose, hydroxyapatite and Affi-Gel Blue column chromatography. The specific activity of the purified enzyme was 91.3 nmol chenodeoxycholic acid formed/min per mg of protein. The reaction was reversible, and the optimum pH of the enzyme for the oxidation was about 8.5, whereas that for the reduction was about 5.0 A molecular weight of the enzyme was estimated to be about 130,000 by Superose 6TM gel filtration chromatography. The apparent Km value for 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid was 35.7 microM and that for NADPH was 90.9 microM. The preferred substrate for the enzyme was 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid rather than 3 alpha,12 alpha-dihydroxy-7-keto-5 beta-cholanoic acid, a 7-keto-bile acid analogue. The enzyme also preferred the unconjugated form to the conjugated forms. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of reduced form of glutathione to the reaction mixture, indicating that the enzyme requires a sulfhydryl group for activity.