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 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.

FIG. 1

(A) Proposed metabolic pathway for DDVA by S. paucimobilis SYK-6. (B) Pathway for the conversion of 2,3-dihydroxybiphenyl (2,3-DHBP) to benzoate by the polychlorinated biphenyl-degrading bacteria. The proposed DDVA metabolic pathway follows the previous one (15). Enzymes: LigZ, OH-DDVA oxygenase; LigAB, protocatechuate 4,5-dioxygenase; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase. TCA, tricarboxylic acid.

FIG. 2

Preparation of DDVA (X), OH-DDVA (VI), and DDPA (IX). Details of the synthesis from vanillic acid ethyl ester (I) are described in Materials and Methods. Et, ethyl; Ac, acetyl.

FIG. 3

Deletion analysis to locate the OH-DDVA oxygenase gene (ligZ). The direction of transcription from the vector-located promoter (Plac) is depicted by a thin arrow. The solid arrow represents the location of the OH-DDVA oxygenase gene (ligZ), and the broken arrow indicates another ORF, which overlaps ligZ. The OH-DDVA oxygenase activities of E. coli MV1190 containing pTE01, pTE491, and the deletion plasmids derived from pTE491 are presented on the right. Activity conferred by pTE491 in the absence of IPTG induction is shown in parentheses.

FIG. 4

Nucleotide and deduced amino acid sequences for the OH-DDVA oxygenase gene (ligZ) from S. paucimobilis SYK-6. The deduced amino acid sequence is presented below the nucleotide sequence. The putative RBS and −10 and −35 promoter sequences are indicated by single underlining and double underlining, respectively. The N-terminal amino acid sequence of the 38-kDa product purified partially from E. coli MV1190 containing pFK09 is indicated by broken underlining. Another in-frame ATG initiation codon at nucleotide position 223 is indicated by dots above the nucleotide sequence.

FIG. 5

SDS-PAGE of OH-DDVA oxygenase preparations. Lanes: 1 and 5, molecular mass standard proteins; 2, crude extract of E. coli MV1190(pFK09); 3, active fraction separated on a Mono Q HR 10/10 column; 4, active fraction separated on a Hi Load 16/60 Superdex 200 preparative-grade column after the Mono Q HR 10/10 column.

FIG. 6

Alignment of amino acid sequences of OH-DDVA oxygenase (LigZ) and the β subunit (LigB) of protocatechuate 4,5-dioxygenase from S. paucimobilis SYK-6. Identical amino acid residues are boxed and shaded. Functionally similar amino acid residues are shaded. A region that is highly conserved between LigZ and LigB is indicated by dots above the LigZ sequence. Asterisks below the LigB sequence represent histidine residues conserved among class III extradiol dioxygenases. Dashes indicate gaps.

FIG. 7

Phylogenetic tree of meta-cleavage enzymes. The phylogenetic tree was constructed by the unweighted pair-group clustering method (29). The enzyme classification of Spence et al. (38) is indicated on the right. Enzymes: LigB, β subunit of protocatechuate 4,5-dioxygenase of SYK-6 (30); CarBb, catalytic subunit of the meta-cleavage enzyme for 2′-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (35); HpcB, 3,4-dihydroxyphenylacetate 2,3-dioxygenase of E. coli C (32); MhpB, 2,3-dihydroxyphenylpropionate 1,2-dioxygenase of E. coli (38); MpcI, catechol 2,3-dioxygenase I (metapyrocatechase I) of A. eutrophus JMP222 (14); BphC LB400, 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas cepacia LB400 (13); BphC KF707, 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707 (7); BphC KKS102, 2,3-dihydroxybiphenyl dioxygenase of Pseudomonas sp. strain KKS102 (18)); XylE DK1, catechol 2,3-dioxygenase of Pseudomonas putida(pDK1) (2); BphC2 P6, 2,3-dihydroxybiphenyl dioxygenase II of R. globerulus P6 (1); BphC3 P6, 2,3-dihydroxybiphenyl dioxygenase III of R. globerulus P6 (1).