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Journal of Bacteriology, December 2000, p. 6950-6957, Vol. 182, No. 24
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.
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
*
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.
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