Open AccessThe 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in Sphingomonas paucimobilis SYK-6
Author(s) -
Hirofumi Hara,
Eiji Masai,
Yoshihiro Katayama,
Masao Fukuda
Publication year - 2000
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.182.24.6950-6957.2000
Subject(s) - sphingomonas paucimobilis , escherichia coli , biology , biochemistry , open reading frame , microbiology and biotechnology , gene , dioxygenase , isoelectric point , molecular mass , enzyme , peptide sequence , bacteria , genetics
Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by theO -demethylase and converted into protocatechuate (PCA) and 3-O -methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kbEco RI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB ), 2-pyrone-4,6-dicarboxylate hydrolase (ligI ), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC ), we found theligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. TheligJ gene is transcribed in the same direction asligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream fromligA . TheligJ gene product (LigJ), expressed inEscherichia coli , was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30°C. TheKm for OMA and theV max were determined to be 138 μM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. TheligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that theligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.