A tetrahydrofolate-dependent O-demethylase, LigM, is crucial for catabolism of vanillate and syringate in Sphingomonas paucimobilis SYK-6.
Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.
Abe
Tomokuni
T
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Masai
Eiji
E
Miyauchi
Keisuke
K
Katayama
Yoshihiro
Y
Fukuda
Masao
M
D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics; D013763:Tetrahydrofolates / Q000378:metabolism*; D014641:Vanillic Acid / Q000378:metabolism*
D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D005707:Gallic Acid; C025525:3-O-methylgallic acid; C001945:syringic acid; D010090:Oxidoreductases, O-Demethylating; D014641:Vanillic Acid
Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347-51
7012838
Methods Enzymol. 1979;68:245-67
232214
J Mol Biol. 1986 May 5;189(1):113-30
3537305
Nucleic Acids Res. 1988 Jan 11;16(1):358
3340535
Nucleic Acids Res. 1988 Aug 11;16(15):7583-600
2970625
J Bacteriol. 1988 Oct;170(10):4924-30
3170489
J Bacteriol. 1990 May;172(5):2704-9
2185230
J Bacteriol. 1991 Dec;173(24):7950-5
1744051
Appl Environ Microbiol. 1992 Mar;58(3):925-31
1575495
Gene. 1994 Jul 22;145(1):69-73
8045426
Appl Environ Microbiol. 1998 Mar;64(3):836-42
9501423
Microbiology. 1998 Apr;144 ( Pt 4):965-73
9579070
Appl Environ Microbiol. 1998 May;64(5):1694-9
9572938
Eur J Biochem. 1998 May 1;253(3):706-11
9654069
Appl Environ Microbiol. 1998 Jul;64(7):2520-7
9647824
J Bacteriol. 1999 Jan;181(1):55-62
9864312
J Bacteriol. 1999 Jun;181(11):3494-504
10348863
Appl Environ Microbiol. 2000 Jun;66(6):2311-7
10831404
J Bacteriol. 2000 Dec;182(23):6651-8
11073908
J Bacteriol. 2000 Dec;182(24):6950-7
11092855
J Bacteriol. 2001 Jun;183(11):3276-81
11344134
J Bacteriol. 2003 Jan;185(1):41-50
12486039
J Bacteriol. 2004 May;186(9):2757-65
15090517
J Bacteriol. 2004 Aug;186(15):4951-9
15262932
Anal Biochem. 1976 May 7;72:248-54
942051
Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7
271968
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648-52
377280
Gene. 1985;33(1):103-19
2985470