Gentisate 1,2-dioxygenase from pseudomonas. Purification, characterization, and comparison of the enzymes from Pseudomonas testosteroni and Pseudomonas acidovorans.

摘要: The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P. acidovorans and testosteroni, the two divergent species of group Pseudomonas. Both enzymes exhibit a 40-fold higher specific activity than previous preparations an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 158,000, respectively). different amino terminal sequences, acid contents, isoelectric points. Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively give complex (S 3/2), showing Fe2+ with coordination sites for exogenous ligands. spectra these complexes are altered uniquely each when bound. This suggests substrate or near shows substrate-iron interactions subtly different. kinetic parameters turnover by nearly identical (kcat/Km 4.3 x 10(6) s-1 M-1). cleave wide range analogs substituted in 3 4 ring position, although at reduced rates relative gentisate. Of enzymes, testosteroni 1,2-dioxygenase exhibits substantially lower kcat/Km values compounds. Evidence both steric electronic substituent effects obtained. In accord results Wheelis et al. (Wheelis, M. L., Palleroni, N. J., Stanier, R. Y. (1967) Arch. Mikrobiol. 59, 302-314), shown be metabolized through pathway, only cleavage dioxygenase induced. contrast, exclusively protocatechuate pathway utilizing 4,5-dioxygenase, coinduced. Growth on 3-O-methylbenzoate 5-O-methylsalicylate result approximately 10-fold increase amount 4,5-dioxygenase. Together, suggest induction may adventitious this function fundamentally metabolic pathways related Pseudomonas species.