J. Biochem. 83, 1125-1134 (1978)
Purification and Properties of a-Hydroxy-7--Carboxymuconic -s-Semialdehyde Dehydrogenase Kiyofumi MARUYAMA,* Nakao ARIGA,* Mitsushige TSUDA,* and Kazuo DEGUCHI** •Faculty of General Education, Gifu University, Nagara, Gifu 502, and "Department of Biochemistry, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173 Received for publication, October 31, 1977
a-Hydroxy-7--carboxymuconic e-semialdehyde (HCMS) dehydrogenase was purified about 180-fold with a yield of 22% from a cell-free extract of Pseudomonas ochraceae. The enzyme was adsorbed tightly on blue dextran, possibly at its coenzyme binding sites. This finding made possible a simple purification step by molecular exclusion chromatography on Sephadex G-200. The enzyme adsorbed on blue dextran was eluted in the void volume and was well separated from contaminating proteins which were eluted at various positions. The purified enzyme migrated as a single band on disc gel electrophoresis. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-200 was 67,000. Polyacrylamide gel electrophoresis of the enzyme after sodium dodecyl sulfate denaturation gave a single band at a position corresponding to a molecular weight of 35,000. The enzyme showed a simple protein absorption in the UV region and no significant absorption in the visible region. The enzyme activity was not affected by various metal ions, metal chelating reagents or reducing reagents, but was strongly inhibited by various sulfhydryl reagents. Both NAD and NADP were found to be efficient coenzymes. Various aldehyde derivatives such as acetaldehyde, propionaldehyde, /i-butyraldehyde, iso-butyraldehyde, succinaldehyde, crotonaldehyde, chloral hydrate, glyoxylic acid, DL-glyceraldehyde, benzaldehyde, salicylaldehyde, glucose, and protocatechualdehyde did not serve as substrates. The stoichiometry of HCMS consumption and coenzyme reduction was approximately 1 : 1 . The reaction product, which appeared to be a-hydroxy-7--carboxymuconic acid, showed an absorption maximum at 311 nm at pH 8.0.
The oxidative metabolism of protocatechuic acid by microorganisms is initiated by aromatic ring fission catalyzed by either protocatechuate 4,5dioxygenase [EC J.13.1.8] or protocatechuate 3,4dioxygenease [EC 1.13.1.3] (/). Protocatechuic Abbreviations: HCMS, cr-hydroxy-7-
Purification and Properties of a-Hydroxy-7--Carboxymuconic -s-Semialdehyde Dehydrogenase Kiyofumi MARUYAMA,* Nakao ARIGA,* Mitsushige TSUDA,* and Kazuo DEGUCHI** •Faculty of General Education, Gifu University, Nagara, Gifu 502, and "Department of Biochemistry, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173 Received for publication, October 31, 1977
a-Hydroxy-7--carboxymuconic e-semialdehyde (HCMS) dehydrogenase was purified about 180-fold with a yield of 22% from a cell-free extract of Pseudomonas ochraceae. The enzyme was adsorbed tightly on blue dextran, possibly at its coenzyme binding sites. This finding made possible a simple purification step by molecular exclusion chromatography on Sephadex G-200. The enzyme adsorbed on blue dextran was eluted in the void volume and was well separated from contaminating proteins which were eluted at various positions. The purified enzyme migrated as a single band on disc gel electrophoresis. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-200 was 67,000. Polyacrylamide gel electrophoresis of the enzyme after sodium dodecyl sulfate denaturation gave a single band at a position corresponding to a molecular weight of 35,000. The enzyme showed a simple protein absorption in the UV region and no significant absorption in the visible region. The enzyme activity was not affected by various metal ions, metal chelating reagents or reducing reagents, but was strongly inhibited by various sulfhydryl reagents. Both NAD and NADP were found to be efficient coenzymes. Various aldehyde derivatives such as acetaldehyde, propionaldehyde, /i-butyraldehyde, iso-butyraldehyde, succinaldehyde, crotonaldehyde, chloral hydrate, glyoxylic acid, DL-glyceraldehyde, benzaldehyde, salicylaldehyde, glucose, and protocatechualdehyde did not serve as substrates. The stoichiometry of HCMS consumption and coenzyme reduction was approximately 1 : 1 . The reaction product, which appeared to be a-hydroxy-7--carboxymuconic acid, showed an absorption maximum at 311 nm at pH 8.0.
The oxidative metabolism of protocatechuic acid by microorganisms is initiated by aromatic ring fission catalyzed by either protocatechuate 4,5dioxygenase [EC J.13.1.8] or protocatechuate 3,4dioxygenease [EC 1.13.1.3] (/). Protocatechuic Abbreviations: HCMS, cr-hydroxy-7-
