Peng X, Egashira T, Hanashiro K, Masai E, Nishikawa S, Katayama Y, Kimbara K, Fukuda M
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Appl Environ Microbiol. 1998 Jul;64(7):2520-7. doi: 10.1128/AEM.64.7.2520-2527.1998.
Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249-5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion.
少动鞘氨醇单胞菌SYK-6将木质素相关的联苯化合物2,2'-二羟基-3,3'-二甲氧基-5,5'-二羧酸联苯（DDVA）通过2,2',3-三羟基-3'-甲氧基-5,5'-二羧酸联苯（OH-DDVA）作为中间体转化为5-羧基香草酸（15）。OH-DDVA的开环是DDVA降解途径中的关键步骤。从黏粒文库中分离出的一个15 kb的EcoRI片段弥补了突变体在OH-DDVA上生长缺陷。亚克隆和缺失分析表明，一个1.4 kb的DNA片段包含负责OH-DDVA开环的基因。鉴定出一个编码334个氨基酸的开放阅读框并命名为ligZ。LigZ推导的氨基酸序列与III类双加氧酶家族有大约18%至约21%的同源性，其中包括SYK-6的原儿茶酸4,5-双加氧酶的β亚基（LigB）（Y. Noda，S. Nishikawa，K.-I. Shiozuka，H. Kadokura，H. Nakajima，K. Yano，Y. Katayama，N. Morohoshi，T. Haraguchi和M. Yamasaki，《细菌学杂志》172:2704 - 2709，1990）、嗜碱产碱菌JMP222的儿茶酚2,3-双加氧酶I（MpcI）（M. Kabisch和P. Fortnagel，《核酸研究》18:3405 - 3406，1990）、假单胞菌属CA10菌株的2'-氨基联苯-2,3-二醇间位裂解酶（CarBb）的催化亚基（S. I. Sato，N. Ouchiyama，T. Kimura，H. Nojiri，H. Yamane和T. Omori，《细菌学杂志》179:4841 - 4849，1997）以及大肠杆菌的2,3-二羟基苯丙酸1,2-双加氧酶（MhpB）（E. L. Spence，M. Kawamukai，J. Sanvoisin，H. Braven和T. D. H. Bugg，《细菌学杂志》178:5249 - 5256，1996）。LigZ作用于OH-DDVA形成的开环产物呈黄色，在455 nm处有最大吸收峰，表明发生间位裂解。因此，得出结论LigZ是一种开环双加氧酶。仅在OH-DDVA和2,2',3,3'-四羟基-5,5'-二羧酸联苯中检测到LigZ活性，且该活性依赖于亚铁离子。
