A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kD...
详细信息
A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37degreesC, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues), showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.
Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-eliminati...
详细信息
Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of beta-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported beta-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of beta-elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the beta-elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.
Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent amino-transferase, has been crystallized in space groups P2(1) and C2. Both crystal forms were obtained at pH 7.3 ...
详细信息
Recombinant 7,8-diaminopelargonic acid synthase from Escherichia coli, a pyridoxal-phosphate-dependent amino-transferase, has been crystallized in space groups P2(1) and C2. Both crystal forms were obtained at pH 7.3 with 21% polyethylene glycol and 10% 2-propanol as precipitants. The cell dimensions were a = 130, b = 57.5, c = 117 Angstrom, beta = 110 degrees for the C2 crystals, and a = 58.4, b = 55.6, c = 121 Angstrom, beta = 96.9 degrees for the P21 crystals, which diffract to at least 2.6 and 2.0 Angstrom resolution, respectively.
The enzyme that catalyzes the reversible conversion of N-acetylglutamic .gamma.-semialdehyde and L-glutamate to .alpha.-N-acetyl-L-ornithine and .alpha.-ketoglutarate, acetylornithine .delta.-trnsaminase [EC 2.6.1.11]...
详细信息
The enzyme that catalyzes the reversible conversion of N-acetylglutamic .gamma.-semialdehyde and L-glutamate to .alpha.-N-acetyl-L-ornithine and .alpha.-ketoglutarate, acetylornithine .delta.-trnsaminase [EC 2.6.1.11], was isolated in homogeneous form and crystallized from both the wild-type and the arginine-inducible strains of E. coli W. The MW of the wild-type transaminase is 119,000 while the MW of the arginine-inducible enzyme is 61,000. However, the arginine-inducible acetylornithine .delta.-transaminase is not a breakdown product of the wild-type, arginine-repressible transaminase. Analysis of crude extracts of the wild-type and arginine-inducible strains by varying the acrylamide concentration in polyacrylamide disc gel electrophoresis showed that arginine-inducible and wild-type transaminases differed in ionic charge. Immunochemical analysis of the 2 transaminases showed that neither enzyme would cross-react with antibodies prepared against its counterpart. Treatment of the 2 enzymes with sodium dodecyl sulfate, followed by disc gel electrophoresis revealed that both transaminases were composed of 31,000-dalton subunits. Tryptic digestion of the 2 transaminases showed that nearly identical peptides were present. The overall data suggest that the wild-type and inducible transaminases were products of 2 different structural genes. The 2 transaminases have different MW, ionic charges and antigenic determinants, but both are composed of similar MW subunits and show a high degree of similarity in amino acid content and peptide composition.
暂无评论