Publications

• Isolation and identification of the reaction product of alpha-hydroxy-gamma-carboxymuconic epsilon-semialdehyde dehydrogenase.

  Maruyama K.

  The reaction product of enzymic dehydrogenation of alpha-hydroxy-gamma-carboxymuconic epsilon-semialdehyde (HCMS) was isolated. The analytical data (elemental analyses, IR spectra, mass spectra, proton NMR spectra, and UV spectra) showed that the product was not alpha-hydroxy-gamma-carboxymuconic ... >> More

  The reaction product of enzymic dehydrogenation of alpha-hydroxy-gamma-carboxymuconic epsilon-semialdehyde (HCMS) was isolated. The analytical data (elemental analyses, IR spectra, mass spectra, proton NMR spectra, and UV spectra) showed that the product was not alpha-hydroxy-gamma-carboxymuconic acid (HCMA), the expected primary product of HCMS dehydrogenation, but a lactone of HCMA. The structure of the lactone was tentatively determined as alpha-pyrone-4,6-dicarboxylic acid. HCMS was converted stoichiometrically to the lactone by the purified NAD(P)-linked HCMS dehydrogenase. The lactone was actively metabolized by a cell-free extract prepared from Pseudomonas ochraceae grown on phthalic acid, suggesting that it may be a metabolic intermediate in the bacterial metabolism of protocatechuic acid. Furthermore, a method for the chemical synthesis of the lactose of HCMA is presented and some of its chemical and biochemical properties are described. << Less

  J Biochem 86:1671-1677(1979) [PubMed] [EuropePMC]

• Purification and properties of 2-pyrone-4,6-dicarboxylate hydrolase.

  Maruyama K.

  A hydrolase which catalyzes specifically the interconversion between 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a sing ... >> More

  A hydrolase which catalyzes specifically the interconversion between 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a single band which was coincident with the enzyme activity. The molecular weight of the enzyme was estimated to be 31,000 by gel filtration on Sephadex G-75 and 33,000 by sodium dodecyl sulfate gel electrophoresis. The isoelectric point of the enzyme was determined to be at pH 5.49 by isoelectric focusing. The enzyme is specific for 2-pyrone-4,6-dicarboxylate, and various other lactones did not serve as substrates. The stoichiometry of 2-pyrone-4,6-dicarboxylate hydrolysis, 4-oxalmesaconate formation and proton production was approximately 1:1:1. The optimum pHs are 8.5 and 6.0 for hydrolysis and synthesis of 2-pyrone-4,6-dicarboxylate, respectively. Km values are 87 and 26 microM for 2-pyrone-4,6-dicarboxylate and 4-oxalmesaconate, respectively. At pH 8.5, the ratio of 4-oxalmesaconate to 2-pyrone-4,6-dicarboxylate at equilibrium is about 2.2. Thiol reagents such as HgCl2 and p-chloromercuribenzoate strongly inhibit the enzyme activity. << Less

  J Biochem 93:557-565(1983) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and properties of alpha-hydroxy-gamma-carboxymuconic epsilon-semialdehyde dehydrogenase.

  Maruyama K., Ariga N., Tsuda M., Deguchi K.

  J Biochem 83:1125-1134(1978) [PubMed] [EuropePMC]

• Interaction of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase with Reactive Blue 2 and related dyes.

  Maruyama K.

  Steady-state kinetic analyses suggest that Pseudomonas ochraceae 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (4-carboxy-2-hydroxy-cis,cis-muconate-6-semialdehyde: NADP+ oxidoreductase [EC 1.2.1.45]) functions through an ordered BiBi mechanism. The enzyme binds one NADP+ molecule per s ... >> More

  Steady-state kinetic analyses suggest that Pseudomonas ochraceae 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (4-carboxy-2-hydroxy-cis,cis-muconate-6-semialdehyde: NADP+ oxidoreductase [EC 1.2.1.45]) functions through an ordered BiBi mechanism. The enzyme binds one NADP+ molecule per subunit with a Kd of 4.8 +/-0.8 microM. The enzyme is adsorbed to a Blue Sepharose CL-6B column and can be eluted therefrom with reagents having high affinity for the enzyme such as NADP+, NAD+, ATP, and Reactive Blue 2. Equilibrium dialysis and difference spectral titration show the binding of four molecules of Reactive Blue 2 per enzyme subunit. Two of these dye molecules show high-affinity binding with a Kd of 0.03 +/-0.02 microM. The resulting 1: 2 enzyme-dye complex can be isolated by gel filtration on Bio-Gel P-6. The kinetic, spectroscopic, and chromatographic properties of the complex indicate that the dye-binding sites are different from the coenzyme binding site. The other two dye molecules, in contrast, bind loosely with a Kd of 0.8 +/-0.5 microM to a site overlapping the coenzyme binding site. This is confirmed by the following findings: NADP+ effectively abolishes the difference spectrum associated with the enzyme-dye binding, and the slope of the double reciprocal plot showing the competitive inhibition of the dye (Ki = 0.20 +/-0.02 microM) with respect to NADP+ linearly depends on the square of the dye concentration. Essentially similar results are also obtained with methoxy Reactive Blue 2 and Reactive Blue 4.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  J Biochem 103:714-721(1988) [PubMed] [EuropePMC]

• 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.

  Masai E., Momose K., Hara H., Nishikawa S., Katayama Y., Fukuda M.

  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 ... >> More

  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. << Less

  J. Bacteriol. 182:6651-6658(2000) [PubMed] [EuropePMC]