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L-Histidine

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Title
Metabolism of zinc, copper and iron as affected by dietary protein, cysteine and histidine.
Author
Snedeker SM; Greger JL
Source
J Nutr, 1983 Mar, 113:3, 644-52
Abstract
Zinc, copper and iron utilization were examined in weanling male rats fed a 45% lactalbumin diet (HPro), a 15% lactalbumin diet (LPro), or 15% lactalbumin diets supplemented with: histidine (LProHis), cysteine (LProCys), or histidine and cysteine (LProHisCys). The histidine content and cysteine content of the supplemented diets were equal to the levels of these amino acids in the HPro diet. Zinc utilization was affected by the levels of protein, cysteine, and, to a lesser extent, histidine in the diets. The apparent absorption of zinc and the levels of zinc in their tibias were greater when the rats were fed the HPro, LProCys or LProHisCys diets rather than the LPro or LProHis diets. Liver copper levels were highest when rats consumed the LPro diet. Tissue levels of iron and fecal losses of iron were not affected by the dietary treatments.

Title
Long-term effects of low histidine intake on men.
Author
Cho ES; Anderson HL; Wixom RL; Hanson KC; Krause GF
Source
J Nutr, 1984 Feb, 114:2, 369-84
Abstract
The purpose of this 85-day study was to investigate the long-term effects of histidine depletion on nitrogen utilization in young adult men. A low nitrogen (6.3 g/day), low histidine (10 mg/day) amino acid diet was fed to seven men for 8 weeks. Mean nitrogen balance became negative at the end of the 8-week period. Free histidine in postabsorptive plasma and 24-hour urine decreased significantly during the first 2 weeks of the depletion and remained low and constant for the remaining 6 weeks. Hemoglobin concentration decreased somewhat, and serum iron concentration increased significantly during histidine depletion. Lean body mass, urinary N'-methylhistidine and total creatinine did not change significantly. On addition of histidine to the low histidine diet for 2 weeks, nitrogen retention became positive, plasma and urinary histidine returned to initial values, serum iron fell, and hemoglobin concentration slowly increased. These parameters remained unchanged in two control men fed the same diet supplemented with histidine (1.05 g/day) for 8 weeks. The results suggest that histidine is indispensable for young men consuming a low nitrogen diet.

Title
Effect of histidine, cysteine, glutathione or beef on iron absorption in humans.
Author
Layrisse M; Martínez Torres C; Leets I; Taylor P; Ramírez J
Source
J Nutr, 1984 Jan, 114:1, 217-23
Abstract
One hundred thirteen subjects participated in iron absorption studies in which histidine, cysteine, reduced glutathione or beef were administered with test meals. Increasing doses of histidine from 416 to 2080 mg did not affect the absorption of 2 mg of corn iron. Reduced glutathione significantly increased the absorption from nonheme and heme iron present in black beans, corn and hemoglobin. Increasing doses of either cysteine or glutathione produce the same trend in the increase of absorption from corn iron as that observed from beef. The results suggest that the effect of cysteine on iron absorption is similar in the peptide form and as a single molecule.

Title
Hydrogen bonding of iron-coordinated histidine in heme proteins.
Author
Rousseau DG; Rousseau DL
Address
AT&T Bell Laboratories, Murray Hill, New Jersey 07974.
Source
J Struct Biol, 1992 Jul, 109:1, 13-7
Abstract
The hydrogen-bonding motifs of the proton on the N delta atom of iron-coordinated histidine residues in heme proteins have been classified into three categories: (1) Those in which the hydrogen-bond acceptor is either an amino acid residue (serine) directly adjacent to the histidine or a carbonyl group of the polypeptide chain less than five residues away from the histidine; (2) those in which the hydrogen-bonding acceptor is a carbonyl group of the polypeptide backbone associated with an amino acid residue 8 to 17 residues away from the histidine; and (3) those in which the hydrogen-bonding acceptor is an exogenous water molecule or an amino acid residue located far from the histidine in the amino acid sequence. Some biological functions are defined by this classification, whereas others span all classes.

Title
Transient Raman study of hemoglobin: structural dependence of the iron-histidine linkage.
Author
Friedman JM; Rousseau DL; Ondrias MR; Stepnoski RA
Source
Science, 1982 Dec, 218:4578, 1244-6
Abstract
Low-frequency resonance Raman spectra of transient hemoglobin species were observed within 10 nanoseconds of photolysis. The Raman frequencies of the iron-proximal histidine stretching mode for transient species having either the R or the T quaternary structure are higher than in the corresponding deoxy species. The observed frequency difference in the iron-histidine mode between the R- and T- state transients indicates that there are quaternary structure-dependent protein forces on the iron-histidine bond in the liganded hemoglobins. These differences are interpreted in terms of changes in the tilt of the histidine with respect to the heme plane.

Title
The histidines of the iron-uptake regulation protein, Fur. Author
Saito T; Duly D; Williams RJ
Address
Inorganic Chemistry Laboratory, University of Oxford, UK.
Source
Eur J Biochem, 1991 Apr, 197:1, 39-42
Abstract
There are 12 histidine residues/molecule in the iron-uptake regulation protein (Fur). Here we examine their pH dependence using proton nuclear magnetic resonance spectroscopy. The histidines have widely spread acid dissociation constants but we can not offer a simple explantation for their complicated behaviour.

Title
Structural comparisons of superoxide dismutases.
Author
Harris JI; Auffret AD; Northrop FD; Walker JE
Source
Eur J Biochem, 1980 May, 106:1, 297-303
Abstract
The amino-terminal sequences of superoxide dismutase isolated from seven microorganisms have been determined. These include the first sequences of enzyme from anaerobic phototrophes. Five enzymes contain iron and two manganese. The enzymes are all related to each other but not to the Cu/Zn family of superoxide dismutases. These sequences, taken with six others from the same family, show that there is no clear distinction in sequence between Fe and Mn types. Moreover it demonstrates a wide variation between enzymes from different bacteria. Also enzymes from anaerobes do not seem to be a particularly closely related group and are not more closely related to each other than to enzymes from aerobes. Two histidine residues are conserved in all proteins and secondary structure predictions suggest they are in close proximity in the same alpha-helix. These residues may provide ligands for the bound metal.

Title
Effect of stabilising amino acids on the digestive absorption of heme and non-heme iron.
Author
Vaghefi N; Guillochon D; Bureau F; Jauzac P; Neuville D; Jacob B; Arhan P; Bouglé D
Address
Laboratoire de physiologie digestive et nutritionnelle, CHU, Caen, France.
Source
Reprod Nutr Dev, 1998 Sep-Oct, 38:5, 559-66
Abstract
We used the Ussing chamber model to study heme iron absorption by rat duodenal mucosa. Heme iron was obtained by enzymic digestion of bovine haemoglobin and concentration of heme (HPH). Its uptake and mucosal transfer was compared to iron gluconate (Gluc), at 100 microM and 1 mM. At 100 microM iron uptake (Qtot), mucosal retention (Qm) and transfer across the mucosa (Qs) was similar for the two sources of iron. Qs was significantly higher at 1 mM for Gluc but not for HPH, and was associated with higher levels of Qm. Addition of L-histidine did not improve iron absorption and indeed it decreased it if iron was provided as Gluc. L-cysteine increased the transfer of iron of both sources. In the in vitro model using rat digestive mucosa, heme iron appeared to be an efficiently used source of iron, which might prevent its accumulation by gut when supplied in excess.

Title
Genetics of hemochromatosis.
Author
Cullen LM; Anderson GJ; Ramm GA; Jazwinska EC; Powell LW
Address
Clinical Sciences Unit, Queensland Institute of Medical Research, Brisbane, Australia.
Source
Annu Rev Med, 1999, 50:, 87-98
Abstract
Hereditary hemochromatosis (HHC) is a common autosomal recessive disorder of iron metabolism that results in progressive iron overload and can be fatal if untreated. The hemochromatosis gene (HFE) was identified by positional cloning in 1996. Two missense mutations have been described in HFE. The majority of HHC patients are homozygous for a cysteine-to-tyrosine substitution (C282Y); however, a small number are homozygous for a histidine-to-aspartic-acid substitution (H63D) or are heterozygous for both of these mutations. Mechanisms by which C282Y and H63D may disrupt the normal functioning of HFE have been suggested, but the role of HFE in the process of normal iron metabolism has yet to be clearly defined.

Title
The construction of metal centers in proteins by rational design.
Author
Hellinga HW
Address
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA. hellinga@linnaeus.biochem.duke.edu
Source
Fold Des, 1998, 3:1, R1-8
Abstract
Metalloprotein properties result from the interplay between coordination requirements of the metal center, protein stability, and modulation of the metal center by the surrounding protein matrix. Simple metal centers, which exercise control over the protein by affecting stability or enzyme activity, have been created by rational design. Complex centers, which require control by the protein matrix, have also been constructed.

Title
The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study. Author
Thompson CP; Grady JK; Chasteen ND
Source
J Biol Chem, 1986 Oct, 261:28, 13128-34
Abstract
Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.

Title
Oral iron, dietary ligands and zinc absorption.
Author
Sandström B; Davidsson L; Cederblad A; Lönnerdal B
Source
J Nutr, 1985 Mar, 115:3, 411-4
Abstract
The effect of iron on zinc absorption in humans was investigated by using 65Zn and whole-body counting after 2 wk. Increasing the molar ratio of ferrous iron (with ascorbic acid) to zinc from 1:1 to 2.5:1 did not affect absorption of zinc from water when given in a fasting state; 59 and 58% was absorbed, respectively. However, at an Fe:Zn ratio of 25:1, zinc absorption from water decreased significantly to 34%. When oral iron in the same ratio to zinc was given with a meal, no inhibitory effect was observed (25, 23 and 22%, respectively). Addition of the zinc ligand, histidine, to the water solution decreased the inhibitory effect of the higher dose of iron, resulting in a zinc absorption of 47%. Two weeks of iron preloading did not affect zinc absorption from water. The results demonstrate that when a multimineral supplement is taken on an empty stomach, excessive iron levels can negatively affect zinc absorption. Intake of the supplement with a meal or with a zinc ligand (such as histidine) may overcome this inhibitory effect.

Title
Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases.
Author
Myllylä R; Günzler V; Kivirikko KI; Kaska DD
Address
Collagen Research Unit, University of Oulu, Finland.
Source
Biochem J, 1992 Sep, 286 ( Pt 3):, 923-7
Abstract
A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.

 

Title
An EXAFS study of the interaction of substrate with the ferric active site of protocatechuate 3,4-dioxygenase.
Author
True AE; Orville AM; Pearce LL; Lipscomb JD; Que L Jr
Address
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Source
Biochemistry, 1990 Dec, 29:48, 10847-54
Abstract
X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Title
Oxygen activating nonheme iron enzymes.
Author
Lange SJ; Que L Jr
Address
Department of Chemistry Center for Metals in Biocatalysis, University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455, USA.
Source
Curr Opin Chem Biol, 1998 Apr, 2:2, 159-72
Abstract
The past year has witnessed significant advances in the study of oxygen-activating nonheme iron enzymes. Thirteen crystal structures of substrate and substrate analog complexes of protocatechuate 3, 4-dioxygenase have revealed intimate details about changes at the enzyme active site during catalysis. Crystallographic data have established a 2-His-1-carboxylate facial triad as a structural motif common to a number of mononuclear nonheme iron enzymes, including isopenicillin N synthase, tyrosine hydroxylase and naphthalene dioxygenase. The first metrical data has been obtained for the high valent intermediates Q and X of methane monooxygenase and ribonucleotide reductase, respectively. The number of enzymes thought to have nonheme diiron sites has been expanded to include alkene monooxygenase from Xanthobacter strain Py2 and the membrane bound alkane hydroxylase from Pseudomonas oleovorans (AlkB). Finally, synthetic complexes have successfully mimicked chemistry performed by both mono- and dinuclear nonheme iron enzymes, such as the extradiol-cleaving catechol dioxygenases, lipoxygenase, alkane and alkene monoxygenases and fatty acid desaturases.

Title
Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.
Author
Annunen P; Helaakoski T; Myllyharju J; Veijola J; Pihlajaniemi T; Kivirikko KI
Address
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland.
Source
J Biol Chem, 1997 Jul, 272:28, 17342-8
Abstract
Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha2beta2 tetramer, the beta subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered alpha(II) subunit from human sources. The mRNA for the alpha(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (alpha(II))2beta2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5-15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed alpha(I)alpha(II)beta2 tetramer. PDI/beta polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/beta polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but the Ki of the former for poly(L-proline) was about 200-1000 times that of the latter. In agreement with this, a minor difference, about 3-6-fold, was found between the two enzymes in the Km values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.

Title
Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe-2S] cluster.
Author
Gurbiel RJ; Ohnishi T; Robertson DE; Daldal F; Hoffman BM
Address
Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
Source
Biochemistry, 1991 Dec, 30:49, 11579-84
Abstract
Electron nuclear double resonance (ENDOR) experiments were performed on 14N (natural abundance) and 15N-enriched iron-sulfur Rieske protein in the ubiquinol-cytochrome c2 oxidoreductase from Rhodobactor capsulatus. The experiments proved that two distinct nitrogenous ligands, histidines, are undoubtedly ligated to the Rieske [2Fe-2S] center. The calculations of hyperfine tensors give values similar but not identical to those of the Rieske-type cluster in phthalate dioxygenase of Pseudomonas cepacia and suggest a slightly different geometry of the iron-sulfur cluster in the two proteins.

Title
Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria.
Author
Britt RD; Sauer K; Klein MP; Knaff DB; Kriauciunas A; Yu CA; Yu L; Malkin R
Address
Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, California 94720.
Source
Biochemistry, 1991 Feb, 30:7, 1892-901
Abstract
Electron spin echo envelope modulation (ESEEM) experiments performed on the Rieske Fe-S clusters of the cytochrome b6f complex of spinach chloroplasts and of the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria show modulation components resulting from two distinct classes of 14N ligands. At the g = 1.92 region of the Rieske EPR spectrum of the cytochrome b6f complex, the measured hyperfine couplings for the two classes of coupled nitrogens are A1 = 4.6 MHz and A2 = 3.8 MHz. Similar couplings are observed for the Rieske centers in the three cytochrome bc1 complexes. These ESEEM results indicate a nitrogen coordination environment for these Rieske Fe-S centers that is similar to that of the Fe-S cluster of a bacterial dioxygenase enzyme with two coordinated histidine ligands [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]. The Rieske Fe-S cluster lacks modulation components from a weakly coupled peptide nitrogen observed in water-soluble spinach ferredoxin. Treatment with the quinone analogue inhibitor DBMIB causes a shift in the Rieske EPR spectrum to g = 1.95 with no alteration in the magnetic coupling to the two nitrogen atoms. However, the ESEEM pattern of the DBMIB-altered Rieske EPR signal shows evidence of an additional weakly coupled nitrogen similar to that observed in the spinach ferredoxin ESEEM patterns.

Title
Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity.
Author
Lamberg A; Pihlajaniemi T; Kivirikko KI
Address
Collagen Research Unit, University of Oulu, Finland.
Source
J Biol Chem, 1995 Apr, 270:17, 9926-31
Abstract
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer in which the alpha subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human alpha subunit individually to serine and expressed the mutant alpha subunits together with the wild-type beta subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Fe2+ to a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Fe2+ binding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.

Title
Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters.
Author
Gurbiel RJ; Batie CJ; Sivaraja M; True AE; Fee JA; Hoffman BM; Ballou DP
Address
Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
Source
Biochemistry, 1989 May, 28:11, 4861-71
Abstract
We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.

Title
The catechol 2,3-dioxygenase gene of Rhodococcus rhodochrous CTM: nucleotide sequence, comparison with isofunctional dioxygenases and evidence for an active-site histidine.
Author
Candidus S; van Pée KH; Lingens F
Address
Institut fÂur Mikrobiologie, UniversitÂat Hohenheim, Stuttgart, FRG.
Source
Microbiology, 1994 Feb, 140 ( Pt 2):, 321-30
Abstract
In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb Bg/II fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a catechol 2,3-dioxygenase (C23O) accepting both 3-methylcatechol and 2,3-dihydroxybiphenyl as substrates could be detected. The plasmid-encoded gene for C23O of R. rhodochrous CTM and its flanking regions were sequenced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The derived amino acid sequence of the C23O was compared to that of nine other enzymes, which all catalyse the extradiol cleavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Gram-positive bacterium. The role of four strongly conserved histidines was examined by chemical modification of the histidyl residues of the native enzyme by diethylpyrocarbonate. For that purpose the C23O was purified to homogeneity from E. coli harbouring pSC1701. However, the enzyme lost its activity during the purification. Activity could partially be restored by treatment with Fe2+ and reducing agents.

Title
Active site structure of Rieske-type proteins: electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter capsulatus and molecular modeling studies of a Rieske center.
Author
Gurbiel RJ; Doan PE; Gassner GT; Macke TJ; Case DA; Ohnishi T; Fee JA; Ballou DP; Hoffman BM
Address
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
Source
Biochemistry, 1996 Jun, 35:24, 7834-45
Abstract
Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [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]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

Title
Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase.
Author
Hurtubise Y; Barriault D; Sylvestre M
Address
INRS-SantÆe, Institut National de la Recherche Scientifique, Pointe-Claire, QuÆebec, H9R 1G6 Canada.
Source
J Biol Chem, 1996 Apr, 271:14, 8152-6
Abstract
Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate. In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.

Title
Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components [published erratum appears in J Bacteriol 1996 Feb;178(3):943]
Author
Hurtubise Y; Barriault D; Powlowski J; Sylvestre M
Address
INRS-SantÆe, Institut National de la Recherche Scientifique, Pointe-Claire, QuÆebec, Canada.
Source
J Bacteriol, 1995 Nov, 177:22, 6610-8
Abstract
In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.

Title
An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.
Author
Kita A; Kita S; Fujisawa I; Inaka K; Ishida T; Horiike K; Nozaki M; Miki K
Address
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Source
Structure, 1999 Jan, 7:1, 25-34
Abstract
BACKGROUND: Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS: The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS: The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.

Title
Cyanide and nitric oxide binding to reduced protocatechuate 3,4-dioxygenase: insight into the basis for order-dependent ligand binding by intradiol catecholic dioxygenases.
Author
Orville AM; Lipscomb JD
Address
Department of Biochemistry, Medical School, and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455-0347, USA. Source
Biochemistry, 1997 Nov, 36:46, 14044-55
Abstract
EPR-silent, chemically reduced protocatechuate 3,4-dioxygenase (Er) binds NO at the active site Fe2+ to yield an EPR-active, S = 3/2 species that blocks subsequent binding of all other exogenous ligands. In contrast, addition of NO to a preformed Er.CN- complex yields an EPR-active, S = 1/2 species [Er.(CN)x.NO] that exhibits resolved superhyperfine splitting from 13CN-, 15/14NO, and a protein-derived 14N. Simulations of the EPR spectra observed for the Er.(CN)x.NO complex formed with 12CN- and 13CN- (1:1) show that CN- binds in two iron ligand sites (x >/= 2). The two cyanides exhibit similar, but distinguishable, hyperfine coupling constants. This demonstrates unambiguously that at least three exogenous ligands (two cyanides and NO) can bind to the Fe2+ simultaneously and strongly suggests that at least one histidine ligand is retained in the complex. The Er.(CN)>/=2.NO complex readily exchanges both of the bound cyanides for the substrate analog, 2-hydroxyisonicotinic acid N-oxide (INO), to form a Er.INO.NO complex exhibiting the same S = 3/2 type EPR spectrum that is observed for this complex in the absence of CN-. Because the dead-end Er.NO complex does not accumulate during the exchange, the results suggest that Er.(CN)>/=2. NO and Er.INO.NO are in conformational states that allow facile exchange of INO and CN- but not NO. The results are interpreted in the context of the known X-ray crystal structures for the ferric form of the resting enzyme (Eox) and numerous Eox.substrate, inhibitor, and CN- complexes, all of which have a charge neutral iron center. It is proposed that the binding of one CN- causes dissociation of an anionic endogenous ligand which begins a series of conformational changes analogous to those initiated by anionic substrate binding to Eox. This results in a new unique coordination site for NO, and a new second site for CN-; both cyanide sites are utilized when the enzyme subsequently binds substrates or INO.

Title
Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases.
Author
Neidle EL; Hartnett C; Ornston LN; Bairoch A; Rekik M; Harayama S
Address
Department of Biology, Yale University, New Haven, Connecticut 06511.
Source
J Bacteriol, 1991 Sep, 173:17, 5385-95
Abstract
The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.

Title
Cloning of the alpha subunit of prolyl 4-hydroxylase from Drosophila and expression and characterization of the corresponding enzyme tetramer with some unique properties.
Author
Annunen P; Koivunen P; Kivirikko KI
Address
Collagen Research Unit, Biocenter and the Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland.
Source
J Biol Chem, 1999 Mar, 274:10, 6790-6
Abstract
Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha2beta2 tetramers, whereas the Caenorhabditis elegans enzyme is an alphabeta dimer, the beta subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha subunit and 31% identity to the C. elegans alpha subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1. 9-kilobase mRNA coding for this alpha subunit was present in Drosophila larvae.

Title
Gentisate 1,2-dioxygenase from Haloferax sp. D1227.
Author
Fu W; Oriel P
Address
Department of Microbiology, Michigan State University, East Lansing 48824-1101, USA.
Source
Extremophiles, 1998 Nov, 2:4, 439-46
Abstract
Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42,000 +/- 1,000 Da subunits, with a native molecular weight of 174,000 +/- 6,000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45 degrees C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified.

 

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