Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights.

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading alpha-ketoglutarate dioxygenase.

The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the alpha-ketoglutarate (alpha-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and alpha-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g-tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of alpha-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding.

Ascorbic acid-dependent turnover and reactivation of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase using thiophenoxyacetic acid.

The first step in catabolism of the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is catalyzed by 2,4-D/alpha-ketoglutarate (alpha-KG)-dioxygenase (TfdA) in Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134. This oxygen- and ferrous-ion-dependent enzyme couples the oxidative decarboxylation of alpha-KG (yielding CO2 and succinate) with the oxidation of 2,4-D to produce 2,4-dichlorophenol and glyoxylate. TfdA was shown to utilize thiophenoxyacetic acid (TPAA) to produce thiophenol, allowing the development of a continuous spectrophotometric assay for the enzyme using the thiol-reactive reagent 4,4'-dithiodipyridine. In contrast to the reaction with 2,4-D, however, the kinetics of TPAA oxidation were nonlinear and ascorbic acid was found to be required for and consumed during TPAA oxidation. The ascorbic acid was needed to reduce a reversibly oxidized inactive state that was formed by reaction of the ferrous enzyme with oxygen, either in the absence of substrate or in the presence of TPAA. The dependency on this reductant was not due to an uncoupling of alpha-KG decarboxylation from substrate hydroxylation, as has been reported for several other alpha-KG-dependent hydroxylases. Significantly, the rate of formation of this reversibly oxidized species was much lower when the enzyme was turning over 2,4-D. Evidence also was obtained for the generation of an inactive enzyme species that could not be reversed by ascorbate. The latter species, not associated with protein fragmentation, arose from an oxidative reaction that is likely to involve hydroxyl radical reactions. On the basis of initial rate studies, the kcat and Km values for TPAA were estimated to be 20-fold lower and 80-fold higher than the corresponding values for 2,4-D. The results are incorporated into a model of TfdA reactivity involving both catalytic and inactivating events.

Frequency of rheumatic diseases in patients with acute anterior uveitis.

The frequency of associated rheumatic diseases was studied in 271 patients with acute anterior uveitis (AAU). In a retrospective examination of 154 patients with AUU (mean follow-up period of 6 years) associated rheumatic symptoms were observed in 64 (41.6%). Forty-one patients (26.6%) had ankylosing spondylitis and 39 (25.3%) manifestations of Reiter's disease. Radiographic sacro-iliitis was seen in 35 (34%) of 103 consecutive x-ray examined patients with AAU. Furthermore, in another series of 38 patients, who all, in addition to having AAU, also complained of low back pain or had manifestations of Reiter's disease, 23 (60.5%) had radiographic sacro-iliitis. Classical ankylosing spondylitis was more frequent in men with AAU whereas milder forms of the disease occurred more equally in both sexes. HLA-B27 occurred in 35 (87.5%) of 40 HLA-typed patients with AAU. Associated rheumatic diseases occurred in 18 (51.4%) of the 35 HLA-B27 positive patients but in none of the HLA-B27 negative patients. The results support the hypothesis that a pleiotropic HLA-B27 associated gene may determine the susceptibility to AAU, sacro-iliitis, ankylosing spondylitis, and Reiter's disease.

Ocular inflammation in Reiter's disease after Salmonella enteritis.

We studied characteristics of ocular inflammation in Reiter's disease after Salmonella enteritis in eight patients. After an acute onset with diarrhea, fever in six patients, and headache in three patients, all patients developed arthritis; six patients had myalgia; six patients, urethritis; and one patient, carditis. Sacroiliitis was found in four patients. All patients had HLA-B28 antigen. Conjunctivitis occurred in seven patients. It was mostly mild with no chemosis, follicles, or keratitis, and resolved in ten days. In one case palpebral edema, chemosis in the conjunctiva, and purulent exudate were seen. One patient had transient episodes of keratitis and corneal erosion for two months and episodes of conjunctivitis for 11 months. Three patients developed unilateral acute anterior uveitis with aqueous flare, cells, fine keratic precipitates, and fibrinous exudation from three to four years after the onset of the illness. One of these patients had vitritis and macular and papillary edema.

Ocular inflammation associated with Yersinia infection.

We studied characteristics of ocular inflammation associated with Yersinia infection in 23 patients. After an acute onset with fever, diarrhea, and abdominal pain, 22 patients developed arthritis, 11 patients developed myalgia, 11 patients developed Reiter's syndrome, 17 patients developed acute anterior uveitis, and nine patients developed conjunctivitis. Sacroiliitis was found in 12 patients. The patients had high erythrocyte sedimentation rates, leukocytosis, and lack of antinuclear antibodies and rheumatoid factor. All 17 patients tested had HLA-B27 antigen. The patients with acute anterior uveitis showed aqueous flare, cells, fine keratic precipitates, and often exudates, posterior synechiae, vitritis, and macular edema. Acute anterior uveitis was mostly unilateral and resolved during corticosteroids on the average during the first six weeks; recurrences were seen in about half of the cases. Conjunctivitis was generally mild with no chemosis, follicles, or keratitis; and it resolved in one week without treatment. Our results indicate that in HLA-B27 positive patients infective agents can trigger acute anterior uveitis or conjunctivitis, which often occur together with rheumatic diseases.