4042-36-8Relevant articles and documents
A colorimetric assay method for measuring D-glutamate cyclase activity
Ariyoshi, Makoto,Hamase, Kenji,Homma, Hiroshi,Katane, Masumi,Matoba, Satoaki,Mita, Masashi,Miyamoto, Tetsuya,Motoda, Risa,Nakayama, Kazuki,Saitoh, Yasuaki,Sakai-Kato, Kumiko,Sekine, Masae,Tateishi, Shuhei
, (2020)
In mammals, metabolism of free D-glutamate is regulated by D-glutamate cyclase (DGLUCY), which reversibly converts D-glutamate to 5-oxo-D-proline and H2O. Metabolism of these D-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-D-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-D-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet–visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of D-glutamate and 5-oxo-D-proline, and cardiac function.
Structural and enzymatic properties of mammalian D-glutamate cyclase
Katane, Masumi,Ariyoshi, Makoto,Tateishi, Shuhei,Koiwai, Sachi,Takaku, Kaoruko,Nagai, Kenichiro,Nakayama, Kazuki,Saitoh, Yasuaki,Miyamoto, Tetsuya,Sekine, Masae,Mita, Masashi,Hamase, Kenji,Matoba, Satoaki,Homma, Hiroshi
, p. 10 - 18 (2018/07/25)
D-Glutamate cyclase (DGLUCY) is a unique enzyme that reversibly converts free D-glutamate to 5-oxo-D-proline and H2O. Mammalian DGLUCY is highly expressed in the mitochondrial matrix in the heart, and its downregulation disrupts D-glutamate and/or 5-oxo-D-proline levels, contributing to the onset and/or exacerbation of heart failure. However, detailed characterisation of DGLUCY has not yet been performed. Herein, the structural and enzymatic properties of purified recombinant mouse DGLUCY were examined. The results revealed a dimeric oligomerisation state, and both D-glutamate-to-5-oxo-D-proline and 5-oxo-D-proline-to-D-glutamate reactions were catalysed in a stereospecific manner. Catalytic activity is modulated by divalent cations and nucleotides including ATP and ADP. Interestingly, the presence of Mn2+ completely abolished the 5-oxo-D-proline-to-D-glutamate reaction but stimulated the D-glutamate-to-5-oxo-D-proline reaction. The optimum pH is ~8.0, similar to that in the mitochondrial matrix, and the catalytic efficiency for D-glutamate is markedly higher than that for 5-oxo-D-proline. These findings suggest that DGLUCY functions as a metalloenzyme that degrades D-glutamate in the mitochondrial matrix in mammalian cells. The results also provide insight into the correlation between DGLUCY enzyme activity and the physiological and pathological roles of D-glutamate and 5-oxo-D-proline in cardiac function, which is of relevance to the risk of onset of heart failure.
Mapping the substrate selectivity of new hydrolases using colorimetric screening: Lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes
Liu, Andrew Man Fai,Somers, Neil A.,Kazlauskas, Romas J.,Brush, Terry S.,Zocher, Frank,Enzelberger, Markus M.,Bornscheuer, Uwe T.,Horsman, Geoff P.,Mezzetti, Alessandra,Schmidt-Dannert, Claudia,Schmid, Rolf D.
, p. 545 - 556 (2007/10/03)
Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed a colorimetric screening method to measure the enantioselectivity of hydrolases. Here we apply this rapid screening method to map the substrate selectivity of four new hydrolases: lipases from the thermophilic Bacillus thermocatenulatus (DSM 730, BTL2) and a filamentous fungus Ophiostoma piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tue 20, SDE). We screened a general library of 29 substrates and a chiral library of 23 pairs of enantiomers. All four hydrolases catalysed the hydrolysis of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified for the solketal esters: BTL2, butyrate, E = 7.9 (R); octanoate, E = 4.9 (R) and 3-bromo-2-methyl propionate methyl esters, PFE, E = 12 (S); SDE, E = 5.6 (S). OPL showed low enantioselectivity toward all substrates tested. The current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).