Title
Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy.
Author
Sono M; Dawson JH
Source
Biochim Biophys Acta, 1984 Sep, 789:2, 170-87
Abstract
In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.

Title
Amino acid sequence, spectral, oxygen-binding, and autoxidation properties of indoleamine dioxygenase-like myoglobin from the gastropod mollusc Turbo cornutus.
Author
Suzuki T; Kawamichi H; Imai K
Address
Laboratory of Biochemistry, Faculty of Science, Kochi University, Japan. suzuki@cc.kochi-u.ac.jp
Source
J Protein Chem, 1998 Nov, 17:8, 817-26
Abstract
Myoglobin was isolated from the radular muscle of the archaeogastropod mollusc Turbo cornutus (Turbinidae). This myoglobin is a monomer carrying one protoheme group; the molecular mass was estimated by SDS-PAGE to be about 40 kDa, 2.5 times larger than that of usual myoglobin. The cDNA-derived amino acid sequence of 375 residues was determined, of which 327 residues were identified directly by chemical sequencing of internal peptides. The amino acid sequence of Turbo myoglobin showed no significant homology with any other usual 16-kDa globins, but showed 36% identity with the myoglobin from Sulculus diversicolor (Haliotiidae) and 27% identity with human indoleamine 2,3-dioxygenase, a tryptophan-degrading enzyme containing heme. Thus, the Turbo myoglobin can be counted among the myoglobins which evolved from the same ancestor as that of indoleamine 2,3-dioxygenase. The absorbance ratio of gamma to CT maximum (gamma/CT) of Turbo metmyoglobin was 17.8, indicating that this myoglobin probably possesses a histidine residue near the sixth coordination position of heme iron. The Turbo myoglobin binds oxygen reversibly. Its oxygen equilibrium properties are similar to those of Sulculus myoglobin, giving P50 = 3.5 mm Hg at pH 7.4 and 20 degrees C. The pH dependence of autoxidation of Turbo oxymyoglobin was quite different from that of mammalian myoglobin, suggesting a unique protein folding around the heme cavity of Turbo myoglobin. A kinetic analysis of autoxidation indicates that the amino acid residue with pKa = 5.4 is involved in the reaction. The autoxidation reaction was enhanced markedly at pH 7.6, but not at pH 5.5 and 6.3 in the presence of tryptophan. We suggest that a noncatalytic binding site for tryptophan, in which several dissociation groups with pKa > or = 7.6 are involved, remains in Turbo myoglobin as a relic of molecular evolution.

Title
Degradation of polychlorinated biphenyl metabolites by naphthalene-catabolizing enzymes.
Author
Barriault D; Durand J; Maaroufi H; Eltis LD; Sylvestre M
Address
INRS-Santé, Université du Québec, Pointe-Claire, Québec, Canada H9R 1G6.
Source
Appl Environ Microbiol, 1998 Dec, 64:12, 4637-42
Abstract
The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1, 2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2, 3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl to 2, 3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3, 4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl (3,4-DH-2,2',5,5'-TCB), respectively. Our data also suggested that purified 1, 2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3, 4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3, 4-DH-2,2',5,5'-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.

Title
Deduced primary structure of rat tryptophan-2,3-dioxygenase.
Author
Maezono K; Tashiro K; Nakamura T
Address
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Source
Biochem Biophys Res Commun, 1990 Jul, 170:1, 176-81
Abstract
The complete amino acid sequence of the tryptophan 2,3-dioxygenase (TO) of rat liver was determined from the nucleotide sequence of a full length TO cDNA isolated from a rat liver cDNA library and determined its primary structure. TO was encoded in a mRNA of about 1.7 kb containing an open reading frame of 1218 bp. According to the deduced amino acid sequence, the monomeric polypeptide of TO consisted of 406 amino acid residues with a calculated molecular weight of 47,796 daltons. It has twelve histidine residues around its hydrophobic region, which has homology with some heme proteins and oxygenase, suggesting that this hydrophobic region might to be the core of TO for the activity.

Title
Site-directed mutagenesis of human lysyl hydroxylase expressed in insect cells. Identification of histidine residues and an aspartic acid residue critical for catalytic activity.
Author
Pirskanen A; Kaimio AM; Myllylä R; Kivirikko KI
Address
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Finland.
Source
J Biol Chem, 1996 Apr, 271:16, 9398-402
Abstract
Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe2+ to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.

Title
Resonance Raman studies of Rieske-type proteins.
Author
Kuila D; Schoonover JR; Dyer RB; Batie CJ; Ballou DP; Fee JA; Woodruff WH
Address
Isotope and Structural Chemistry Division, Los Alamos National Laboratory, NM 87545.
Source
Biochim Biophys Acta, 1992 Dec, 1140:2, 175-83
Abstract
Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.

Title
The electron-transport proteins of hydroxylating bacterial dioxygenases.
Author
Mason JR; Cammack R
Address
Division of Biosphere Sciences, King's College London, United Kingdom.
Source
Annu Rev Microbiol, 1992, 46:, 277-305
Abstract
The degradation of aromatic compounds by aerobic bacteria frequently begins with the dihydroxylation of the substrate by nonheme iron-containing dioxygenases. These enzymes consist of two or three soluble proteins that interact to form an electron-transport chain that transfers electrons from reduced nucleotides (NADH) via flavin and [2Fe-2S] redox centers to a terminal dioxygenase. The dioxygenases may be classified in terms of the number of constituent components and the nature of the redox centers. Class I consists of two-component enzymes in which the first protein is a reductase containing both a flavin and a [2Fe-2S] redox center and the second component is the oxygenase; Class II consists of three-component enzymes in which the flavin and [2Fe-2S] redox centers of the reductase are on a separate flavoprotein and ferredoxin, respectively; and Class III consists of three-component enzymes in which the reductase contains both a flavin and [2Fe-2S] redox center but also requires a second [2Fe-2S] center on a ferredoxin for electron transfer to the terminal oxygenase. Further subdivision is based on the the type of flavin (FMN or FAD) in the reductase, the coordination of the [2Fe-2S] center in the ferredoxin, and the number of terminal oxygenase subunits. From the deduced amino acid sequence of several dioxygenases the ligands involved in the coordination of the nucleotides, iron-sulfur centers, and mononuclear nonheme iron active site are proposed. On the basis of their spectroscopic properties and unusually high redox potentials, the [2Fe-2S] clusters of the ferredoxins and terminal oxygenases have been assigned to the class of Rieske-type iron-sulfur proteins. The iron atoms in the Rieske iron-sulfur cluster are coordinated to the protein by two histidine nitrogens and two cysteine sulfurs.

Title
Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.
Author
Lawrence CC; Sobey WJ; Field RA; Baldwin JE; Schofield CJ
Address
Dyson Perrins Laboratory, Oxford, UK.
Source
Biochem J, 1996 Jan, 313 ( Pt 1):, 185-91
Abstract
Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Title
Evidence of histidine coordination to the catalytic ferrous ion in the ring-cleaving 2,2',3-trihydroxybiphenyl dioxygenase from the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1.
Author
Bertini I; Capozzi F; Dikiy A; Happe B; Luchinat C; Timmis KN
Address
Department of Chemistry, University of Florence, Italy.
Source
Biochem Biophys Res Commun, 1995 Oct, 215:3, 855-60
Abstract
The 1H NMR spectra of an aromatic ring-cleaving extradiol dioxygenase, 2,2',3-trihydroxybiphenyl dioxygenase of the dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1, are reported. In the catalytically active reduced form of the monomeric enzyme (MW = 32 kDa), three broad strongly downfield shifted signals were observed, two of which disappeared in D2O solution. Their shifts and linewidths are consistent with ring NH and meta-like protons of coordinated histidines. These signals show strong sensitivity to the presence of the substrate. The oxidized form of the enzyme shows no hyperfine shifted signals. It is suggested that the high spin Fe(II) ion present in the active form of the enzyme is coordinated by at least two histidines. This is the first report of hyperfine shifted NMR signals being detected for an extradiol dioxygenase.

Title
Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.
Author
Spence EL; Kawamukai M; Sanvoisin J; Braven H; Bugg TD
Address
Department of Chemistry, University of Southampton, United Kingdom.
Source
J Bacteriol, 1996 Sep, 178:17, 5249-56
Abstract
The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.

Title
Coordination of the Rieske-type [2Fe-2S] cluster of the terminal iron-sulfur protein of Pseudomonas putida benzene 1,2-dioxygenase, studied by one- and two-dimensional electron spin-echo envelope modulation spectroscopy.
Author
Shergill JK; Joannou CL; Mason JR; Cammack R
Address
Center for the Study of Metals in Biology and Medicine, King's College, University of London, U.K.
Source
Biochemistry, 1995 Dec, 34:51, 16533-42
Abstract
One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei [N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [14N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds. For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871] indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.

Title
Human 4-hydroxyphenylpyruvate dioxygenase. Primary structure and chromosomal localization of the gene.
Author
Rüetschi U; Dellsén A; Sahlin P; Stenman G; Rymo L; Lindstedt S
Address
Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Sweden.
Source
Eur J Biochem, 1993 May, 213:3, 1081-9
Abstract
We report the primary structure of 4-hydroxyphenylpyruvate dioxygenase [4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver lambda gt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino-acid sequence. These peptide sequences covered 86% of the cDNA-derived amino-acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa. There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C-terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4-hydroxyphenylpyruvate dioxygenases. The identity of the C-terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i.e. amino acids which have been implicated as ligands for iron in 2-oxoacid-dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14-->qter by Southern-blot analysis of human-rodent somatic-cell hybrids.

Title
Molecular characterization of a gene encoding a homogentisate dioxygenase from Aspergillus nidulans and identification of its human and plant homologues.
Author
Fernández Cañón JM; Peñalva MA
Address
Departamento de MicrobiologÆia Molecular, Centro de Investigaciones BiolÆogicas del CSIC, Madrid, Spain.
Source
J Biol Chem, 1995 Sep, 270:36, 21199-205
Abstract
We report here the first characterization of a gene encoding a homogentisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA protein catalyzes an essential step in phenylalanine catabolism, and disruption of the gene results in accumulation of homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (Mr = 50,168) containing 21 histidine and 23 tyrosine residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the affinity-purified protein has homogentisate dioxygenase activity. A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E. (1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.

Title
Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.
Author
Jiang H; Parales RE; Lynch NA; Gibson DT
Address
Department of Microbiology, The University of Iowa, Iowa City, 52242, USA.
Source
J Bacteriol, 1996 Jun, 178:11, 3133-9
Abstract
The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.

Title
Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad.
Author
Han S; Eltis LD; Timmis KN; Muchmore SW; Bolin JT
Address
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
Source
Science, 1995 Nov, 270:5238, 976-80
Abstract
Polychlorinated biphenyls (PCBs) typify a class of stable aromatic pollutants that are targeted by bioremediation strategies. In the aerobic degradation of biphenyl by bacteria, the key step of ring cleavage is catalyzed by an Fe(II)-dependent extradiol dioxygenase. The crystal structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase from a PCB-degrading strain of Pseudomonas cepacia has been determined at 1.9 angstrom resolution. The monomer comprises amino- and carboxyl-terminal domains. Structural homology between and within the domains reveals evolutionary relationships within the extradiol dioxygenase family. The iron atom has five ligands in square pyramidal geometry: one glutamate and two histidine side chains, and two water molecules.

Title
Purification and characterization of 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase.
Author
Fukumori F; Hausinger RP
Address
Center for Microbial Ecology, Michigan State University, East Lansing 48824.
Source
J Biol Chem, 1993 Nov, 268:32, 24311-7
Abstract
The Alcaligenes eutrophus 2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase, encoded by the tfdA gene of plasmid pJP4, is an Fe(II)-dependent enzyme that catalyzes the conversion of 2,4-dichlorophenoxyacetate to 2,4-dichlorophenol and glyoxylate concomitant with the decomposition of alpha-ketoglutarate to form succinate and carbon dioxide (Fukumori, F., and Hausinger, R. P. (1993) J. Bacteriol. 175, 2083-2086). Using recombinant Escherichia coli cells that overexpress the tfdA gene, the thermolabile enzyme (stable only up to 30 degrees C) was purified to apparent homogeneity (specific activity of 16.9 mumol of substrate converted min-1 mg of protein-1) by a simple two-step procedure. The native protein has an apparent M(r) of 50,000 +/- 2,500, consistent with a homodimeric structure. Ferrous ion is absolutely required for activity and cannot be replaced by several other divalent cations tested. Ascorbic acid stimulates dioxygenase activity and reduces the rate of enzyme inactivation by a metal ion-mediated process. The enzyme exhibits maximum activity at pH 6.5-7, however, it is stable over a pH range of 6.5-11. Although capable of hydroxylating a wide range of phenoxyacetates and related compounds, the enzyme exhibits the greatest affinity (Km 17.5 +/- 1.0 microM) and highest catalytic efficiency for 2,4-dichlorophenoxyacetate. Similarly, alpha-ketoglutarate is the preferred co-substrate (Km 3.20 +/- 0.54 microM) for the enzyme, but it can utilize a range of other alpha-ketoacids with lower efficiency. Results from chemical modification studies are consistent with the presence of multiple essential histidine residues in the enzyme.

Title
Characterization of the first enzyme in 2,4-dichlorophenoxyacetic acid metabolism.
Author
Hausinger RP; Fukumori F
Address
Department of Microbiology, Michigan State University, East Lansing 48824, USA.
Source
Environ Health Perspect, 1995 Jun, 103 Suppl 5:, 37-9
Abstract
This paper reviews the properties of the Alcaligenes eutrophus JMP134 tfdA gene product, the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation. The gene was overexpressed in Escherichia coli and several of its enzymatic properties were characterized. Although this enzyme catalyzes a hydroxylation reaction, it is not a monooxygenase. Rather, TfdA is an Fe(II) and alpha-ketoglutarate-dependent dioxygenase that metabolizes the latter cosubstrate to succinate and carbon dioxide. A variety of other phenoxyacetates and alpha-ketoacids can be used by the enzyme, but the greatest catalytic efficiencies were found using 2,4-D and alpha-ketoglutarate. The enzyme possesses multiple essential histidine residues, whereas catalytically essential cysteine and lysine groups do not appear to be present.

Title
A fully active catalytic domain of bovine aspartyl (asparaginyl) beta-hydroxylase expressed in Escherichia coli: characterization and evidence for the identification of an active-site region in vertebrate alpha-ketoglutarate-dependent dioxygenases.
Author
Jia S; McGinnis K; VanDusen WJ; Burke CJ; Kuo A; Griffin PR; Sardana MK; Elliston KO; Stern AM; Friedman PA
Address
Merck Research Laboratories, West Point, PA 19486.
Source
Proc Natl Acad Sci U S A, 1994 Jul, 91:15, 7227-31
Abstract
The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl) beta-hydroxylase (EC 1.14.11.16) specifically hydroxylates one aspartic or asparagine residue in certain epidermal growth factor-like domains of a number of proteins. The expression in Escherichia coli, purification, characterization of a fully active catalytic domain, and evidence for the identification of an active-site region of this enzyme are described. Sequence alignment analyses among the vertebrate alpha-ketoglutarate-dependent dioxygenases and chemical modification studies were undertaken aimed at locating specific regions of 52-kDa recombinant aspartyl (asparaginyl) beta-hydroxylase involved in substrate binding and/or catalysis. Based upon these studies, an alignment of the C-terminal regions of prolyl and lysyl hydroxylase and of aspartyl (asparaginyl) beta-hydroxylase is proposed. When histidine-675, an invariant residue located in a region of homology within this alignment, was mutated to an alanine residue in aspartyl (asparaginyl) beta-hydroxylase (H675A), no enzymatic activity was detected. Chemical modification studies show that the wild-type protein is protected from iodo[14C]acetamide labeling by Fe2+/alpha-ketoglutarate whereas the H675A mutant protein is not, suggesting that this mutant does not bind Fe2+/alpha-ketoglutarate.

Title
gamma-Butyrobetaine hydroxylase. Structural characterization of the Pseudomonas enzyme.
Author
Rüetschi U; Nordin I; Odelhög B; Jörnvall H; Lindstedt S
Address
Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Sweden.
Source
Eur J Biochem, 1993 May, 213:3, 1075-80
Abstract
gamma-Butyrobetaine hydroxylase is a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of gamma-butyrobetaine to carnitine, the last step in the biosynthesis of carnitine from lysine. The primary structure of the enzyme from Pseudomonas sp. AK1 has been determined. Sequence analysis of the intact protein and of peptides from essentially three different digests established the presence of a peptide chain containing 383 residues, and an N-terminal truncated form of 382 residues. The two chains have molecular masses of 43,321 Da and 43,207 Da, respectively, and are identical except for the presence or absence of an N-terminal asparagine residue; the shorter form starts with an alanine residue. In preparations of the dimeric protein, the two chains occur in an approximate ratio of 1:1. There are nine cysteine residues and 13 histidine residues, i.e. amino acids which have been postulated as ligands for iron binding. In spite of functional similarities, there appears to be no clear sequence similarities with any of the other mammalian 2-oxoglutarate-dependent dioxygenases so far characterized. ------------------

Title
The core metal-recognition domain of MerR.
Author
Zeng Q; StÁlhandske C; Anderson MC; Scott RA; Summers AO
Address
The Department of Microbiology, University of Georgia, Athens 30602, USA.
Source
Biochemistry, 1998 Nov, 37:45, 15885-95
Abstract
MerR, the metalloregulatory protein of the mercury-resistance operon (mer) has unusually high affinity and specificity for ionic mercury, Hg(II). Prior genetic and biochemical evidence suggested that the protein has a structure consisting of an N-terminal DNA binding domain, a C-terminal Hg(II)-binding domain, and an intervening region involved with communication between these two domains. We have characterized a series of MerR deletion mutants and found that as little as 30% of the protein (residues 80-128) forms a stable dimer and retains high affinity for Hg(II). Biophysical measures indicate that this minimal Hg(II)-binding domain assumes the structural characteristics of the wild-type full-length protein both in the Hg(II) center itself and in an immediately adjacent helical protein domain. Our observations are consistent with the core Hg(II)-binding domain of the MerR dimer being constituted by a pair of antiparallel helices (possibly in a coiled-coil conformation) comprised of residues cysteine 82 through cysteine 117 from each monomer followed by a flexible loop through residue cysteine 126. These antiparallel helices would have a potential Hg(II)-binding site at each end. However, just as in the full-length protein, only one of these potential binding sites in the deleted proteins actually binds Hg(II). Title
A carbon-13 NMR comparative study of metal ion substitutions in human carbonic anhydrase I carboxymethylated at active-site histidine-200.
Author
Khalifah RG; Morley PJ
Source
Arch Biochem Biophys, 1984 Aug, 232:2, 632-9
Abstract
Human carbonic anhydrase I (EC 4.2.1.1), the low-activity isozyme, has a reactive active-site Histidine-200 that is known to be specifically modified at N tau with haloacetates. Using [1-13C]bromoacetate, we previously introduced a highly sensitive 13C NMR probe into the active site of the enzyme and studied the interaction of the carboxymethyl carboxylate with the active-site zinc, as well as the ionization properties of the carboxymethylated histidine-200 side chain. In the present work, these studies have been extended to metalloderivatives of the enzyme in which the intrinsic zinc has been replaced by Cd2+, Hg2+, and Co2+. In the former two metals, spin-1/2 isotopes (113Cd and 199Hg) in the absence of inhibitory halides were utilized to search for two-bond spin-spin couplings in the spectrum of the 13C-enriched carboxymethyl carboxylate under conditions where coordination exists in the Zn and Co derivatives. The absence of splittings and titration studies of the chemical shift of the resonance both established the absence of coordination. The pH dependence of the carboxylate, which reflects the ionization of the CmHis-200 ring, was observed in the presence and absence of bound inhibitors. Marked differences were seen among the four metalloderivatives in all these properties, suggesting great sensitivity of the active site to the nature of the metal inserted. The data suggest extreme caution in extrapolating results from metal ion substitution studies to the native zinc enzyme, and may reflect functional significance of this sensitivity in the catalysis.

Title
Biotoxicity of mercury to Chlorella vulgaris as influenced by amino acids.
Author
Mohapatra DK; Mohanty L; Mohanty RC; Mohapatra PK
Address
Department of Botany, Uktal University, Bhubaneswar, India.
Source
Acta Biol Hung, 1997, 48:4, 497-504
Abstract
The toxicity of mercury ion, on Chlorella vulgaris, is largely influenced by amino acids. Five amino acids, namely alanine, asparagine, glutamic acid, cysteine and histidine, were added separately to the medium containing static dose of mercury. Survival (%) of the alga was reduced with the increasing concentrations of mercury. Of these five amino acids, cysteine was found to be the most effective while alanine and glutamic acid were the least effective on reducing the toxic effect of mercury on the alga measured in terms of growth, chlorophyll and protein content. The order of detoxification was Alanine < Glutamate < Asparargine < Histidine < Cysteine. Amino acids from ligands with Hg2+ making it less toxic to the alga and produce an additional source of energy for growth and development.

Title
Biotoxicity of mercury as influenced by mercury(II) speciation.
Author
Farrell RE; Germida JJ; Huang PM
Address
Department of Soil Science, University of Saskatchewan, Saskatoon, Canada.
Source
Appl Environ Microbiol, 1990 Oct, 56:10, 3006-16
Abstract
Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Title
Modulation of mu, delta and kappa opioid receptors in rat brain by metal ions and histidine.
Author
Tejwani GA; Hanissian SH
Address
Department of Pharmacology, College of Medicine, Ohio State University, Columbus 43210-1239.
Source
Neuropharmacology, 1990 May, 29:5, 445-52
Abstract
The effect of zinc (Zn2+) and several other trace elements was studied on the binding of the opioid receptor agonists [3H] DAGO [( ([Tyr-D-Ala-Gly-Methyl-Phe-Glyol]-enkephalin)a, [3H] DSTLE ([Tyr-D-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H] EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC50 (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the Bmax (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations.(ABSTRACT TRUNCATED AT 250 WORDS)