Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6.
Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilis SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (ligC). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that ligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. The ligC gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of ligC showed 19 to 20% identity with 3-chlorobenzoate cis-dihydrodiol dehydrogenase of Alcaligenes sp. strain BR60 and phthalate cis-dihydrodiol dehydrogenases of Pseudomonas putida NMH102-2 and Burkholderia cepacia DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13-56, 1994). The ligC gene was expressed in Escherichia coli and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP(+) by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25 degrees C, respectively. The K(m) for NADP(+) was estimated to be 24.6 +/- 1.5 microM, which was approximately 10 times lower than that for NAD(+) (252 +/- 3.9 microM). The K(m)s for CHMS in the presence of NADP(+) and NAD(+) are 26.0 +/- 0.5 and 20.6 +/- 1.0 microM, respectively. Disruption of ligC in S. paucimobilis SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the ligC insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of ligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.
Masai
E
E
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan. emasai@vos.nagaokaut.ac.jp
Momose
K
K
Hara
H
H
Nishikawa
S
S
Katayama
Y
Y
Fukuda
M
M
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D004269:DNA, Bacterial; D049308:Dioxygenases; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000378:metabolism*; D011753:Pyrones / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D011753:Pyrones; C024153:alpha-pyrone-4,6-dicarboxylic acid; C021389:2-hydroxy-4-carboxymucconate-6-semialdehyde dehydrogenase; D010105:Oxygenases; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; D000445:Aldehyde Oxidoreductases