A novel 2-aminomuconate deaminase in the nitrobenzene degradation pathway of Pseudomonas pseudoalcaligenes JS45.
2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The Km for 2-aminomuconate was approximately 67 microM, and the Vmax was 125 micromol x min(-1) x mg(-1). The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.
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Air Force Research Laboratory, Tyndall Air Force Base, Florida 32403, USA.
Spain
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JC
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000627:Aminophenols / Q000378:metabolism; D001711:Biotransformation; D002396:Catechols / Q000378:metabolism; D003437:Crotonates / Q000378:metabolism; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D017421:Sequence Analysis; D013379:Substrate Specificity
D000627:Aminophenols; D002396:Catechols; D003437:Crotonates; D009578:Nitrobenzenes; C027667:2-aminophenol; C002788:oxalocrotonate; D000619:Aminohydrolases; C110186:2-aminomuconate deaminase; C034221:catechol