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

• Published in: Applied and Environmental Microbiology 1998-07, Vol.64 (7), p.2520-2527
• Main Authors: Peng, X. (Nagaoka University of Technology, Nagaoka, Niigata, Japan.), Egashira, T, Hanashiro, K, Masai, E, Nishikawa, S, Katayama, Y, Kimbara, K, Fukuda, M
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; AMINO ACID SEQUENCES; Amino acids; AROMATIC COMPOUNDS; Bacterial Proteins; Base Sequence; BIODEGRADACION; BIODEGRADATION; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Biphenyl Compounds - metabolism; CHEMICAL COMPOSITION; Cloning; COMPOSE AROMATIQUE; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; COMPUESTOS AROMATICOS; Enzymes; ENZYMIC ACTIVITY; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; GENBANK/AB007823; Genetics; Genetics and Molecular Biology; Lignin - metabolism; MICROBIAL DEGRADATION; Microbiology; Mission oriented research; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; OXIDOREDUCTASES; OXIDORREDUCTASAS; OXYDOREDUCTASE; Oxygenases - genetics; Phylogeny; PSEUDOMONAS; SECUENCIA NUCLEOTIDICA; Sequence Alignment; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Substrate Specificity; Transformation, Bacterial; Zymomonas - enzymology; Zymomonas - genetics
• ISSN: 0099-2240; 1098-5336
• 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'-dicarboxy-biphenyl (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