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

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan.
Journal of Bacteriology (Impact Factor: 2.81). 01/2001; 182(23):6651-8. DOI: 10.1128/JB.182.23.6651-6658.2000
Source: PubMed


Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of Sphingomonas paucimobilisSYK-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 thatligC was located 295 bp downstream of ligB, which encodes the large subunit of the PCA 4,5-dioxygenase. TheligC 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 ofAlcaligenes sp. strain BR60 and phthalatecis-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 inEscherichia 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°C, respectively. TheKm
for NADP+ was estimated to be 24.6 ± 1.5 μM, which was approximately 10 times lower than that for NAD+ (252 ± 3.9 μM). TheKm
s for CHMS in the presence of NADP+ and NAD+ are 26.0 ± 0.5 and 20.6 ± 1.0 μM, respectively. Disruption of ligC inS. 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 ofligC into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for ligAB gene expression.

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