Publications (8)44.76 Total impact
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Article: Crystal structure of human intrinsic factor: cobalamin complex at 2.6-A resolution.
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ABSTRACT: The structure of intrinsic factor (IF) in complex with cobalamin (Cbl) was determined at 2.6-A resolution. The overall fold of the molecule is that of an alpha(6)/alpha(6) barrel. It is a two-domain protein, and the Cbl is bound at the interface of the domains in a base-on conformation. Surprisingly, two full-length molecules, each comprising an alpha- and a beta-domain and one Cbl, and two truncated molecules with only an alpha- domain are present in the same asymmetric unit. The environment around Cbl is dominated by uncharged residues, and the sixth coordinate position of Co(2+) is empty. A detailed comparison between the IF-B12 complex and another Cbl transport protein complex, trans-Cbl-B12, has been made. The pH effect on the binding of Cbl analogues in transport proteins is analyzed. A possible basis for the lack of interchangeability of human and rat IF receptors is presented.Proceedings of the National Academy of Sciences 11/2007; 104(44):17311-6. · 9.68 Impact Factor -
Article: Crystal structure at 2.5 A resolution of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli.
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ABSTRACT: Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, utilizing molecular oxygen as a terminal electron acceptor. To accomplish this transformation, CAOs utilize two cofactors: a mononuclear copper, and a unique redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ or TOPA quinone). TPQ is derived via posttranslational modification of a specific tyrosine residue within the protein itself. In this study, the structure of an amine oxidase from Hansenula polymorpha has been solved to 2.5 A resolution, in which the precursor tyrosine is unprocessed to TPQ, and the copper site is occupied by zinc. Significantly, the precursor tyrosine directly ligands the metal, thus providing the closest analogue to date of an intermediate in TPQ production. Besides this result, the rearrangement of other active site residues (relative to the mature enzyme) proposed to be involved in the binding of molecular oxygen may shed light on how CAOs efficiently use their active site to carry out both cofactor formation and catalysis.Biochemistry 09/2000; 39(32):9709-17. · 3.42 Impact Factor -
Article: Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1)).
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ABSTRACT: Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.Biochemistry 08/2000; 39(26):7678-88. · 3.42 Impact Factor -
Article: Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).
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ABSTRACT: Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.Acta Crystallographica Section D Biological Crystallography 12/1999; 55(Pt 11):1933-6. · 12.62 Impact Factor -
Article: Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin.
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ABSTRACT: The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.Biochemistry 01/1999; 37(49):17128-36. · 3.42 Impact Factor -
Article: Enzymatic and electron transfer activities in crystalline protein complexes.
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ABSTRACT: Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme.Journal of Biological Chemistry 05/1996; 271(16):9177-80. · 4.77 Impact Factor -
Article: Crystallization and preliminary crystallographic studies of human beta-glucuronidase.
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ABSTRACT: Crystals of human beta-glucuronidase have been obtained by the vapor diffusion method, using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the orthorhombic space group P222(1), with cell dimensions a = 134.5 A, b = 95.1 A, c = 124.4 A. The unit cell contains two copies of the tetrameric enzyme. Complete native data have been collected to a resolution of 2.6 A.Journal of Molecular Biology 10/1993; 233(1):173-6. · 4.00 Impact Factor -
Article: Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin.
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ABSTRACT: The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.Biochemistry 07/1992; 31(21):4959-64. · 3.42 Impact Factor
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1999–2000
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Washington University in St. Louis
- Department of Biochemistry and Molecular Biophysics
Saint Louis, MO, USA
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