Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme

Abstract

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 β 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. 179:48414849, 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.