Molecular cloning and homology modeling of protocatechuate 3,4-dioxygenase from Pseudomonas marginata.
The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas marginata genomic library. These genes pcaG and pcaH, were found when screening the library for hydrolase genes. The two open reading frames of the PCD genes could be identified adjacent to an esterase gene by sequence homology. A 1.7-kb KpnI/ApaI fragment, carrying pcaG and pcaH, was subcloned and the genes were functionally expressed in Escherichia coli. The deduced amino acid sequence shows high homology to previously determined amino acid sequences of bacterial protocatechuate 3,4-dioxygenases. A homology model based on the available crystal structure of the protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa shows high similarity with the binding and catalytic sites.
Petersen
E
E
Institute of Biotechnology, University of Technology Graz, Austria.
Zuegg
J
J
Ribbons
D W
DW
Schwab
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016366:Open Reading Frames; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011520:Protocatechuate-3,4-Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D011520:Protocatechuate-3,4-Dioxygenase; D004950:Esterases