Junker F, Field J A, Bangerter F, Ramsteiner K, Kohler H P, Joannou C L, Mason J R, Leisinger T, Cook A M
Microbiology Institute, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):429-36. doi: 10.1042/bj3000429.
2-Aminobenzenesulphonic acid (2AS) is degraded by Alcaligenes sp. strain O-1 via a previously detected but unidentified intermediate. A mutant of strain O-1 was found to excrete this intermediate, which was isolated and identified by m.s., 1H- and 13C-n.m.r. as 3-sulphocatechol (3SC). Proteins from cell extracts of strain O-1 were separated by anion-exchange chromatography. A multicomponent oxygenase was observed to convert 1 mol each of NADH, O2 and 2AS into 1 mol each of 3SC, NH3 and NAD+. The enzyme presumably catalysed formation of the ring of a 2-amino-2,3-diol moiety, and elimination in the amino group led to a rearomatization. 3SC was further degraded via meta ring cleavage, which could be prevented by inactivation of the 3-sulphocatechol-2,3-dioxygenase (3SC23O) with 3-chlorocatechol. In Tris buffer, the separated 3SC23O catalysed the reaction of 1 mol each of 3SC and O2 involving a transient yellow intermediate, and release of 1 mol of sulphite and two organic products. The major product was identified by n.m.r. and by g.c./m.s. as 5-carboxypenta-2,4-dien-5-olide (CPDO), an indicator of formation of 2-hydroxymuconic acid (2HM). The second product was identified as the Z,E isomer of 2HM by comparison with authentic material. When the CPDO in the product mixture was chemically hydrolysed to (Z,E)-2HM, 1 mol of (Z,E)-2HM/mol of 3SC was observed. If oxygenation of 3SC by 3SC23O was carried out in phosphate buffer, only a single product was detected, a keto form of 2HM. This dioate was also formed from authentic (Z,E)-2HM in phosphate buffer. Formation of the natural product (Z,E)-2HM from the xenobiotic, 3SC, seems to involve oxygenation to the unstable 2-hydroxy-6-sulphonomuconic acid semialdehyde, which hydrolyses spontaneously to 2HM. There would appear to be at least one spontaneous reaction per enzyme reaction in this pathway.
2-氨基苯磺酸（2AS）可被产碱杆菌属菌株O-1降解，降解过程中会经过一个先前已检测到但未鉴定的中间体。研究发现菌株O-1的一个突变体可分泌该中间体，通过质谱、1H-和13C-核磁共振对其进行分离和鉴定，确定为3-磺酸基儿茶酚（3SC）。采用阴离子交换色谱法对菌株O-1细胞提取物中的蛋白质进行分离。观察到一种多组分加氧酶可将1摩尔的NADH、O2和2AS分别转化为1摩尔的3SC、NH3和NAD+。该酶可能催化了2-氨基-2,3-二醇部分环的形成，氨基的消除导致了再芳构化。3SC通过间位环裂解进一步降解，用3-氯儿茶酚使3-磺酸基儿茶酚-2,3-双加氧酶（3SC23O）失活可阻止这种降解。在Tris缓冲液中，分离得到的3SC23O催化1摩尔的3SC和O2反应，反应过程中有一个短暂的黄色中间体，释放出1摩尔的亚硫酸盐和两种有机产物。通过核磁共振和气相色谱/质谱鉴定出主要产物为5-羧基-2,4-二烯-5-内酯（CPDO），它是2-羟基粘康酸（2HM）形成的指示剂。通过与标准物质比较，鉴定出第二种产物为2HM的Z,E异构体。当产物混合物中的CPDO化学水解为（Z,E）-2HM时，观察到每摩尔3SC生成1摩尔（Z,E）-2HM。如果在磷酸盐缓冲液中用3SC23O对3SC进行氧化，只检测到一种产物，即2HM的酮式结构。在磷酸盐缓冲液中，由标准的（Z,E）-2HM也可形成这种二氧代物。从外源性物质3SC形成天然产物（Z,E）-2HM似乎涉及氧化生成不稳定的2-羟基-6-磺酸基粘康酸半醛，该半醛会自发水解为2HM。在这条途径中，每个酶促反应似乎至少有一个自发反应。
