Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0016632 (Fox)
 1,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

T4MOC is a 12.3 kDa soluble Rieske ferredoxin that is obligately required for electron transfer between the oxidoreductase and diiron hydroxylase components of toluene 4-monooxygenase from Pseudomonas mendocina KR1. Our preliminary 1H NMR studies of oxidized and reduced T4MOC [Markley, J. L., Xia, B., Chae, Y. K., Cheng, H., Westler, W. M., Pikus, J. D., and Fox, B. G. (1996) in Protein Structure Function Relationships (Zaidi, Z., and Smith, D., Eds.) pp 135-146, Plenum Press, London] revealed the presence of hyperfine-shifted 1H resonances whose short relaxation times made it impractical to use nuclear Overhauser effect (NOE) measurements for assignment purposes. We report here the use of selective isotopic labeling to analyze the hyperfine-shifted 1H, 2H, and 15N signals from T4MOC. Selective deuteration led to identification of signals from the four Hbeta atoms of cluster ligands C45 and C64 in the oxidized and reduced forms of T4MOC. In the reduced state, the Curie temperature dependence of the Hbeta protons corresponded to that predicted from the simple vector spin-coupling model for nuclei associated with the localized ferric site. The signal at 25.5 ppm in the 1H spectrum of reduced T4MOC was assigned on the basis of selective 2H labeling to the His Hepsilon1 atom of one of the cluster ligands (H47 or H67). This assignment was corroborated by a one bond 1H-13C correlation (at 25.39 ppm 1H and 136.11 ppm 13C) observed in spectra of [U-13C]T4MOC with a 1H-13C coupling constant of approximately 192 Hz. The carbon chemical shift and one bond coupling constant are those expected for 1Hepsilon1-13Cepsilon1 in the imidazolium ring of histidine and are inconsistent with values expected for cysteine 1Halpha-13Calpha. The His Hepsilon1 proton exhibited weak Curie temperature dependence from 283 to 303 K, contrary to the anti-Curie temperature dependence predicted from the spin coupling model for nuclei associated with the localized ferrous site. A 1H peak at -12.3 ppm was observed in spectra of reduced T4MOC; this signal was found to correspond to a hydrogen (probably in an H-bond to the cluster) that exchanged with solvent with a half-time of about 2 days in the oxidized state but with a much longer (undetectable) half-time in the reduced state. These results with T4MOC call into question certain 1H assignments recently reported on the basis of NOE measurements for the comparable Rieske ferredoxin component of an evolutionarily related alkene monooxygenase from Xanthobacter sp. Py2 [Holz, R. C., Small, F. J., and Ensign, S. A, (1997) Biochemistry 36, 14690-14696]. Selective 15N labeling was used to identify hyperfine-shifted 15N NMR signals from the backbone nitrogens of all four cluster ligands (C45, H47, C64, and H67), from the Nepsilon2 atoms of the two histidine ligands (H47 and H67), and from nonligand Gln and Ala residues (Q48 and A66) present in the cluster-binding motif of T4MOC in the oxidized and reduced states. The results indicate that the Ndelta1 of each of the two ligand histidines of T4MOC are ligated to an iron atom and reveal a pattern of H-bonding to the Rieske [2Fe-2S] center involving four (H47, Q48, A66, and H67 of T4MOC) of the five backbone amide H-bonds expected on the basis of comparison with the crystal structures of other related Rieske proteins; the fifth backbone amide (I50 of T4MOC) failed to exhibit a hyperfine shift. This anomaly may arise from the lack of an associated disulfide in T4MOC, a fundamental structural difference between the three types of Rieske proteins that may be related to functional diversity in this protein family.
 ...
PMID:Detection and classification of hyperfine-shifted 1H, 2H, and 15N resonances of the Rieske ferredoxin component of toluene 4-monooxygenase. 988 13

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.
 ...
PMID:Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis. 1065 45

Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
 ...
PMID:Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone. 1523 3

It is useful to have available a variety of catchment-scale water quality models that range in complexity, spatial resolution and data requirements. In a previous paper [Warren, C., Mackay, D., Whelan, M., Fox, K., 2005. Mass balance modelling of contaminants in river basins: a flexible matrix approach. Chemosphere 61, 1458-1467] a series of simple to intermediately complex mass balance models was presented which can be used for tiered exposure assessments in river basins. The connectivity of the segments is expressed using a matrix that permits flexibility in application, enabling the model to be re-segmented and applied to different catchments as required. In this paper, the intermediate models, QWASI matrix-rate constant (QMX-R) and QWASI matrix-fugacity (QMX-F) are used to estimate concentrations of linear alkylbenzene sulfonates (LAS) in the rivers Aire and Calder, UK, and of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Fraser River basin, Canada. The results compare satisfactorily with monitoring data, suggesting that these QWASI-based models for exposure and risk assessment may be applicable under data-limited conditions. The use of QWASI-based models for regulatory purposes in an evaluative river system is also discussed with reference to assessments of para-dichlorobenzene (pDCB), trichloroethylene (TCE), bis(2-ethylhexyl) phthalate (DEHP) and toluene. It is shown that multi-media QWASI model predictions can be usefully depicted graphically on chemical space diagrams and used to highlight regions in which advection, partitioning to sediments and volatilization may be important determinants of chemical fate in river systems.
 ...
PMID:Mass balance modelling of contaminants in river basins: application of the flexible matrix approach. 1736 5