Candida delta-aminovalerate: alpha-ketoglutarate aminotransferase: purification and enzymologic properties

A new enzyme that catalyzes the transamination of delta-aminovalerate with alpha-ketoglutarate was purified to homogeneity from adapted cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 118,000. The transaminase behaved as a dimer with two similar subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a maximum activity in the pH range of 7.8-8.5 and at 40 degrees C. alpha-Ketoglutarate and to a lesser extent pyridoxal 5'-phosphate were effective protecting agents toward temperature raising. The enzyme exhibits absorption maximum at 330 and 410 nm. The enzyme catalyzes the transamination between omega-amino acids and alpha-ketoglutarate. delta-Aminovaleric acid is the best amino donor. The Km values for delta-aminovalerate, alpha-ketoglutarate, and pyridoxal 5'-phosphate determined from the Lineweaver-Burk plot were 4.9 mM, 3.6 mM, and 22.7 microM, respectively. The inhibitory effect of various amino acids analogues on the transamination reaction between delta-aminovalerate and alpha-ketoglutarate was studied, and Ki values were determined.     

Purification and properties of beta-alanine aminotransferase from rabbit liver

beta-Alanine aminotransferase from rabbit liver has been purified 1,700-fold over the initial liver extract. The purified enzyme was shown to be homogeneous by disc electrophoresis and SDS polyacrylamide electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 95,000 +/- 5,700 and the subunit molecular weight was 48,000 +/- 2,100. The enzyme showed absorption maxima at 282, 330, and 414 nm and contained only 1 mol of pyridoxal 5'-phosphate/mol of dimer. The pH optimum for enzyme activity was 8.8 and the Km values for beta-alanine and 2-oxoglutaric acid were calculated to be 3.9 and 1.4 mM, respectively. The enzyme catalyzed transamination of various omega-amino acids with 2-oxoglutaric acid, which was a favourable amino acceptor. beta-Alanine, gamma-aminobutyric acid, and beta-aminoisobutyric acid, which are naturally occurring substrates, were preferred amino donors, but taurine, alanine, ornithine, spermine, and spermidine were not. 6-Azauracil inhibited the enzyme activity with a Ki of approximately 1.5 mM. From the above properties, beta-alanine aminotransferase from rabbit liver was seen to closely resemble with 4-aminobutyrate aminotransferase from liver and brain.     

The primary structure of omega-amino acid:pyruvate aminotransferase.

The complete amino acid sequence of bacterial omega-amino acid:pyruvate aminotransferase (omega-APT) was determined from its primary structure. The enzyme protein was fragmented by CNBr cleavage, trypsin, and Staphylococcus aureus V8 digestions. The peptides were purified and sequenced by Edman degradation. omega-ATP is composed of four identical subunits of 449 amino acids each. The calculated molecular weight of the enzyme subunit is 48,738 and that of the enzyme tetramer is 194,952. No disulfide bonds or bound sugar molecules were found in the enzyme structure, although 6 cysteine residues were determined per enzyme subunit. Sequence homologies were found between an omega-aminotransferase, i.e. mammalian and yeast ornithine delta-aminotransferases, fungal gamma-aminobutyrate aminotransferase and 7,8-diaminoperalgonate aminotransferase, and 2,2-dialkylglycine decarboxylase. The enzyme structure is not homologous to those of aspartate aminotransferases (AspATs) including the enzymes of Escherichia coli and Sufolobus salfactaricus, though significant homology in the three-dimensional structures around the cofactor binding site has been found between omega-APT and AspATs (Watanabe, N., Sakabe, K., Sakabe, N., Higashi, T., Sasaki, K., Aibara, S., Morita, Y., Yonaha, K., Toyama, S., and Fukutani, H. (1989) J. Biochem. 105, 1-3).     

Purification and properties of a novel enzyme,N-acylamino acid racemase,from Streptomyces atratus Y-53

A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (M_r) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal M_r. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (K_m) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co²⁺, Mg²⁺ and Mn²⁺, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase). Correspondence to: S. Tokuyama     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

Preliminary crystallographic study of omega-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126.

Large single crystals of ω-amino acid: pyruvate aminotransferase, were prepared by dialysis of the enzyme solution against 2.2 m-ammonium sulphate solution at pH 7.8. X-ray diffraction patterns show that the crystals belong to the orthorhombic space group I222 or I2₁2₁2₁ with unit cell dimensions $\text{a = 124.1}\text{A}\text{?}\text{, b = 137.9}\text{A}\text{?}\text{, and}\text{c = 61.2}\text{A}\text{?}$. The asymmetric unit consists of one monomer of molecular weight 43,000.     

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.     

Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes.

We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.     

Candida L-norleucine,leucine:2-oxoglutarate aminotransferase. Purification and properties

A new enzyme which catalyzes the transamination of L-norleucine (2-aminohexanoic acid) and L-leucine with 2-oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100,000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51,000). The enzyme has a maximum activity in the pH range of 8.0-8.5 and at 55 degrees C. 2-Oxoglutarate, and to a lesser extent pyridoxal 5'-phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L-Norleucine, and L-leucine to a lesser extent, are the best amino donors with 2-oxoglutarate as amino acceptor. The Km values for L-norleucine, L-leucine and 2-oxoglutarate determined from the Lineweaver-Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping-pong bi-bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L-norleucine and L-leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L-norleucine and 2-oxoglutarate was studied and Ki values were determined.     

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.     

Purification and characterization of a novel extracellular Streptomyces lividans 66 enzyme inactivating fusidic acid.

The wild-type strain Streptomyces lividans 66 is resistant against the steroid-like antibiotic fusidic acid. Comparative studies of the wild-type strain and a fusidic acid-sensitive mutant allowed the identification of an extracellular enzyme which inactivates fusidic acid. With the help of a combination of ultrafiltration and chromatographies with Phenyl-Sepharose and an anion exchanger, the enzyme was highly purified. Its apparent molecular mass is 48 kDa, its optimal activity ranges between 45 and 55 degrees C, and its optimal pH is 6.0 to 9.0. It is stimulated by neither monovalent nor divalent ions. The enzyme acts as a specific esterase which removes the acetyl group at C-16 from fusidic acid. The resulting intermediate is unstable, and spontaneous lactonization between C-21 and C-16 occurs rapidly.     

Purification and properties of a xylanase from Streptomyces lividans.

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).