Cloning and characterization of the genes encoding enzymes for the protocatechuate meta-degradation pathway of Pseudomonas ochraceae NGJ1.
The 2-pyrone-4,6-dicarboxylate lactonase gene (proL), the protocatechuate 4,5-dioxygenase alpha and beta subunits genes (proOa and proOb), and the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (proD) were cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1. These genes were in the order proLOaObD on the DNA, and a possible transcription terminator sequence followed. The proL and proD genes were over-expressed in Escherichia coli, and their gene products were purified for identification, while the expression of proOaOb was at a lower level. The protocatechuate meta-degradation operon was reconstituted with the recombinant plasmids and expressed successfully in E. coli.
Maruyama
Kiyofumi
K
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. maruyama@cc.gifu-u.ac.jp
Shibayama
Tomohiko
T
Ichikawa
Aki
A
Sakou
Yukari
Y
Yamada
Satsue
S
Sugisaki
Hiroyuki
H
D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D062385:Hydroxybenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D009876:Operon; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D004269:DNA, Bacterial; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C009091:protocatechuic acid; C021389:2-hydroxy-4-carboxymucconate-6-semialdehyde dehydrogenase; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; D000445:Aldehyde Oxidoreductases