Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases.
In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.
Nogales
Juan
J
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain.
Canales
Angeles
A
Jiménez-Barbero
Jesús
J
García
José Luis
JL
Díaz
Eduardo
E
D000595:Amino Acid Sequence; D001560:Benzidines / Q000737:chemistry; D002374:Catalase / Q000737:chemistry; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D003001:Cloning, Molecular; D003373:Coumaric Acids / Q000494:pharmacology; D049308:Dioxygenases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005707:Gallic Acid / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016958:Pseudomonas putida / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D020578:Sphingomonas / Q000201:enzymology; D013379:Substrate Specificity; D013997:Time Factors; D014641:Vanillic Acid / Q000494:pharmacology
D001560:Benzidines; D003373:Coumaric Acids; D007211:Indoles; C029876:benzidine; D005707:Gallic Acid; C012381:indole-3-carbaldehyde; D002392:Catechin; C004999:ferulic acid; C045651:epigallocatechin gallate; C016517:indole-3-carbinol; D002374:Catalase; D009195:Peroxidase; D010105:Oxygenases; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; C439587:extradiol dioxygenase; D014641:Vanillic Acid