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Journal of Bacteriology, December 2000, p. 6950-6957, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in Sphingomonas paucimobilis SYK-6

Hirofumi Hara,1 Eiji Masai,1,* Yoshihiro Katayama,2 and Masao Fukuda1

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,1 and Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509,2 Japan

Received 26 June 2000/Accepted 21 September 2000

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 the O-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-kb EcoRI 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 the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC 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 from ligA. The ligJ gene product (LigJ), expressed in Escherichia 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. The Km for OMA and the Vmax 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. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ 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.

* Corresponding author. Mailing address: Department of Bioengineering, Nagaoka University of Technology, Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9428. Fax: 81-258-47-9450. E-mail: emasai{at}vos.nagaokaut.ac.jp.

Journal of Bacteriology, December 2000, p. 6950-6957, Vol. 182, No. 24
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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