Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6.
Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.
Peng
Xue
X
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Masai
Eiji
E
Kitayama
Hirotaka
H
Harada
Kyo
K
Katayama
Yoshihiro
Y
Fukuda
Masao
M
D001426:Bacterial Proteins; D001547:Benzaldehydes / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017666:Deuterium Oxide / Q000378:metabolism; D004926:Escherichia coli; D008031:Lignin / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D010795:Phthalic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity; D014641:Vanillic Acid / Q000031:analogs & derivatives / Q000378:metabolism*
C466307:5-carboxyvanillic acid; D001426:Bacterial Proteins; D001547:Benzaldehydes; D010795:Phthalic Acids; D011994:Recombinant Proteins; C050236:dehydrodivanillin; D008031:Lignin; C113517:2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl oxygenase; D010105:Oxygenases; C466306:5-carboxyvanillate decarboxylase; D002262:Carboxy-Lyases; D014641:Vanillic Acid; D017666:Deuterium Oxide