He Z, Spain J C
Air Force Research Laboratory, Tyndall Air Force Base, Florida 32403, USA.
J Bacteriol. 1998 May;180(9):2502-6. doi: 10.1128/JB.180.9.2502-2506.1998.
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.
2-氨基粘康酸是哺乳动物色氨酸代谢的中间产物，也是假产碱假单胞菌JS45对硝基苯生物降解的中间产物。菌株JS45将2-氨基粘康酸水解为4-草酰巴豆酸，并释放出氨，氨作为微生物生长的氮源。作为研究这种新型脱氨机制的第一步，我们在此报告2-氨基粘康酸脱氨酶的纯化及一些性质。在变性条件下，纯化后的酶在15%聚丙烯酰胺凝胶电泳中迁移为一条分子量为16.6 kDa的单带。通过凝胶过滤和4%至20%梯度非变性聚丙烯酰胺凝胶电泳估计天然酶的分子量为100 kDa，这表明该酶由六个相同的亚基组成。该酶在室温下稳定，在pH 6.6时表现出最佳活性。2-氨基粘康酸的Km约为67 μM，Vmax为125 μmol·min⁻¹·mg⁻¹。该酶的N端氨基酸序列与数据库中的任何序列均无明显相似性。纯化后的酶将2-氨基粘康酸直接转化为4-草酰巴豆酸，而不是2-羟基粘康酸，这表明脱氨是通过亚胺中间体进行的。
