Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H 4 folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H 4 folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H 4 folate, forming 5-methyl-H 4 folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H 4 folate. The possible role of ligH is discussed.Lignin is the most abundant aromatic compound in nature, and the utilization of lignin for production of chemicals has been expected. One of the practical procedures for utilizing lignin is its conversion to valuable intermediate metabolites using the microbial lignin degradation enzyme systems (22). It is known that the degradation of native lignin is initiated by the attack by lignin peroxidase, manganese peroxidase, and laccase secreted by white rot fungi (14), and bacteria contribute to the process of mineralization of the abundant lignin-derived compounds found in soil (44, 47). In microbial degradation of lignin-derived compounds, vanillate and syringate are the important intermediate metabolites. Sphingomonas paucimobilis SYK-6 is able to utilize these compounds and various ligninderived biaryls as the sole source of carbon and energy (20-22, 29, 30). Vanillate and syringate are O demethylated by this strain to produce protocatechuate (PCA) and 3-O-metylgallate (3MGA), respectively. PCA is further degraded through the PCA 4,5-cleavage pathway. In contrast, it has been found that 3MGA is degraded via multiple pat...