Lignin is the most abundant aromatic compound on the earth, and its mineralization is a fundamental step in the terrestrial carbon cycle. It is expected that lignin can be used as biomass by converting it to valuable materials. Bacterial enzyme systems for lignin degradation and modification are of great use for this purpose. Sphingomonas paucimobilis
SYK-6 is able to grow on various dimeric lignin compounds, including β-aryl ether, biphenyl, and diarylpropane, as sole carbon and energy sources (18
). We have characterized the enzymes and genes involved in β-aryl ether cleavage (16
) and biphenyl degradation (23
), which include essential and late limiting steps of lignin degradation, respectively. These unique specific lignin degradation enzymes in SYK-6 would be suitable tools for conversion of lignin to useful intermediate metabolites.
Vanillate and syringate are important intermediate metabolites from lignin, having guaiacyl and syringyl moieties, respectively. In SYK-6, vanillate and syringate are converted to protocatechuate (PCA) and 3-O
-methylgallate (3MGA), respectively, by the O
-demethylase encoded by ligH
). PCA is a key intermediate metabolite among various aromatic degradation pathways. Three kinds of dioxygenases are involved in the aromatic ring cleavage of PCA: PCA 3,4-dioxygenase (5
), PCA 4,5-dioxygenase (22
), and PCA 2,3-dioxygenase (35
). In the case of SYK-6, PCA is metabolized through the PCA 4,5-cleavage pathway (Fig. ), which was enzymatically characterized in 1980s by Kersten et al.
) and Maruyama and colleagues (11–15). PCA is initially transformed to 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) by PCA 4,5-dioxygenase (LigAB). CHMS is nonenzymatically converted to an intramolecular hemiacetal form and then oxidized by CHMS dehydrogenase (11
). The resulting intermediate, 2-pyrone-4,6-dicarboxylate (PDC), is hydrolyzed by PDC hydrolase to yield the keto form and enol form (4-carboxy-2-hydroxymuconate) of 4-oxalomesaconate (OMA), which are in equilibrium (8
). OMA is converted to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) by OMA hydratase (13
). Finally, CHA is cleaved by CHA aldolase to produce pyruvate and oxaloacetate (14
). We previously characterized the PCA 4,5-dioxygenase gene (ligAB
) and PDC hydrolase gene (ligI
); recently, the CHMS dehydrogenase gene (ligC
) was also characterized (E. Masai, K. Momose, H. Hara, S. Nishikawa, Y. Katayama, and M. Fukuda, submitted for publication). However, the PCA 4,5-cleavage pathway has not been genetically characterized in detail.
FIG. 1 The proposed degradation pathway of vanillate and syringate via the PCA 4,5-cleavage pathway in S. paucimobilis SYK-6. LigA and LigB, the small and large subunits of PCA 4,5-dioxygenase (4,5-PCD) (22); LigH, a gene product essential for vanillate and (more ...)
On the other hand, the pathway for 3MGA degradation is ambiguous. PCA 4,5-dioxygenase was reported to catalyze the ring cleavage of 3MGA to form PDC, and metabolism of 3MGA through the PCA 4,5-cleavage pathway was suggested (8
). However, two mutant strains of SYK-6, in which the ligAB
genes were insertionally inactivated could grow on syringate but not on vanillate (19; H. Aoshima, E. Masai, S. Nishikawa, Y. Katayama, and M. Fukuda, Abstr. 8th Int. Symp. Microb. Ecol., abstr. 93, 1998). These results indicated that 3MGA generated from syringate is predominantly metabolized via a pathway other than the PCA 4,5-cleavage pathway.
In this study, we characterized the OMA hydratase gene and the enzymatic properties of the gene product to obtain detailed genetic information on the PCA 4,5-cleavage pathway and insight into the metabolism of syringate in SYK-6. We also present evidence that the OMA hydratase gene is essential to the metabolism of both vanillate and syringate and that OMA is the common intermediate metabolite of vanillate and syringate.