A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate.
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.
Masai
Eiji
E
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan. emasai@vos.nagaokaut.ac.jp
Sasaki
Miyuki
M
Minakawa
Yasunori
Y
Abe
Tomokuni
T
Sonoki
Tomonori
T
Miyauchi
Keisuke
K
Katayama
Yoshihiro
Y
Fukuda
Masao
M
D001483:Base Sequence; D003001:Cloning, Molecular; D003640:Dealkylation; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020578:Sphingomonas / Q000235:genetics / Q000254:growth & development*; D013763:Tetrahydrofolates / Q000378:metabolism*
D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D005707:Gallic Acid; C025525:3-O-methylgallic acid; D010090:Oxidoreductases, O-Demethylating; C023284:vanillate O-demethylase