Abstract
The 3-hydroxybenzoate inducible gentisate 1,2-dioxygenases have been purified to homogeneity from P. acidovorans and P. testosteroni, the two divergent species of the acidovorans group of Pseudomonas. Both enzymes exhibit a 40-fold higher specific activity than previous preparations and have an (alpha Fe)4 quaternary structure (holoenzyme Mr = 164,000 and 158,000, respectively). The enzymes have different amino terminal sequences, amino acid contents, and isoelectric points. Each enzyme contains essential active site iron that is EPR silent but binds nitric oxide quantitatively to give an EPR active complex (S = 3/2), showing that the iron is Fe2+ with coordination sites for exogenous ligands. The EPR spectra of these complexes are altered uniquely for each enzyme when gentisate is bound. This suggests that substrate binds to or near the iron and shows that the substrate-iron interactions of each enzyme are subtly different. The kinetic parameters for turnover of gentisate by the enzymes are nearly identical (kcat/Km = 4.3 x 10(6) s-1 M-1). Both enzymes cleave a wide range of gentisate analogs substituted in the 3 or 4 ring position, although at reduced rates relative to gentisate. Of the two enzymes, P. testosteroni gentisate 1,2-dioxygenase exhibits substantially lower kcat/Km values for the turnover of these compounds. Evidence for both steric and electronic substituent effects is obtained. In accord with the results of Wheelis et al. (Wheelis, M. L., Palleroni, N. J., and Stanier, R. Y. (1967) Arch. Mikrobiol. 59, 302-314), 3-hydroxybenzoate is shown to be metabolized by P. acidovorans through the gentisate pathway, and gentisate 1,2-dioxygenase is the only ring cleavage dioxygenase induced. In contrast, 3-hydroxybenzoate is metabolized by P. testosteroni exclusively through the protocatechuate pathway utilizing protocatechuate 4,5-dioxygenase, although gentisate 1,2-dioxygenase is coinduced. Growth of P. testosteroni on 3-O-methylbenzoate or 5-O-methylsalicylate is shown to result in a approximately 10-fold increase in the amount of gentisate 1,2-dioxygenase relative to protocatechuate 4,5-dioxygenase. Together, these results suggest that induction of gentisate 1,2-dioxygenase by 3-hydroxybenzoate in P. testosteroni may be adventitious and that this enzyme may function in fundamentally different metabolic pathways in the two related Pseudomonas species.
Highlights
3-hydroxybenzoate induced cultures of P. testosteroni contained high levels of protocatechuate 4,5dioxygenase, none was detected in the purified gentisate 1,2-dioxygenase by either activity assay or SDS-PAGE
The amino terminal sequences of the Pseudomonas gentisate 1,2dioxygenases were determined by automated Edman degradation (Table II)
Anaerobic addition of nitric oxide (NO) to gentisate 1,2dioxygenase elicited an EPR signal with features at g -4 and g = 2 (Fig. 2, solid lines), which are characteristic of an S = 3/~spin system originating from the interaction of high spin Fe’+ with the unpaired spin of NO
Summary
Purification of Gentisate 1,2-Dioxygenases-Enzyme were carried out at 4 “C except where indicated. The combined supernatants were heated gradually in two batches to 60 ‘C in a 65 “C water bath, immediately cooled to 15 “C with a dry ice/isopropyl alcohol slurry, and centrifuged at 8875 X g for. The gentisate 1,2dioxygenase activity pool was made 0.5 M in ammonium sulfate and loaded onto a pre-equilibrated column The column was washed with 100 ml of 0.25 M ammonium sulfate in buffer. The activity peak from this column was concentrated as above but with several cycles of dilution with buffer not containing Fe’+ and cysteine. The resuspended pellet from a 33-40% ammonium sulfate fractionation of the resultant supernatant was diluted and loaded onto a pre-equilibrated 14 cm). The pooled activity peak was brought to 0.5 M in ammonium sulfate and loaded onto a column of phenylSepharose CL-4B (2.5 x 8 cm).
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