Characterization of the gallate dioxygenase gene: three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6.
Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase beta and alpha subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K(m) for gallate and the V(max) were determined to be 66.9 +/- 9.3 microM and 42.7 +/- 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.
Kasai
Daisuke
D
Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Masai
Eiji
E
Miyauchi
Keisuke
K
Katayama
Yoshihiro
Y
Fukuda
Masao
M
D003001:Cloning, Molecular; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics*; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D020578:Sphingomonas / Q000201:enzymology / Q000235:genetics*
D005707:Gallic Acid; C001945:syringic acid; D049308:Dioxygenases