Affiliation:
1. Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata
2. Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan
Abstract
ABSTRACT
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 (H
4
folate)-dependent syringate
O
-demethylase gene (
desA
). The
metF
and
ligH
genes, which are thought to be involved in H
4
folate-mediated C
1
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 H
4
folate; 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
120 articles.
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