The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein–ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-­PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

/pdf/prodrg-a-tool-for-high-throughput-crystallography-of-protein-2o0obb3hfy.pdf

PRODRG: a tool for high-throughput crystallography of protein–ligand complexes

https://www.cell.com/trends/plant-science/pdf/S1360-1385(02)02251-3.pdf

GATEWAY vectors for Agrobacterium-mediated plant transformation.

Publisher Summary This chapter investigates the anatomy and taxonomy of protein structures. A protein is a polypeptide chain made up of amino acid residues linked together in a definite sequence. Amino acids are “handed,” and naturally occurring proteins contain only L-amino acids. A simple mnemonic for that purpose is the “corncrib.” The sequence of side chains determines all that is unique about a particular protein, including its biological function and its specific three-dimensional structure. The major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. The analysis and discussion of protein structure is based on the results of three-dimensional X-ray crystallography of globular proteins. The basic elements of protein structures are discussed. The most useful level at which protein structures are to be categorized is the domain, as there are many cases of multiple-domain proteins in which each separate domain resembles other entire smaller proteins. The simplest type of stable protein structure consists of polypeptide backbone wrapped more or less uniformly around the outside of a single hydrophobic core. The outline of the taxonomy is also provided in the chapter.

The anatomy and taxonomy of protein structure.

Prediction of the secondary structure of proteins from their amino acid sequence.

Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.

Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.

/pdf/the-structure-and-properties-of-methylenetetrahydrofolate-44t1jiicwb.pdf

The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.

Thioredoxin reductase two modes of catalysis have evolved.

Two X-ray structures of cobalamin (B12) bound to proteins have now been determined. These structures reveal that the B12 cofactor undergoes a major conformational change on binding to the apoenzymes of methionine synthase and methylmalonyl–coenzyme A mutase: The dimethylbenzimidazole ligand to the cobalt is displaced by a histidine residue from the protein. Two methyltransferases from archaebacteria that catalyze methylation of mercaptoethanesulfonate (coenzyme M) during methanogenesis have also been shown to contain histidine-ligated cobamides. In corrinoid iron-sulfur methyltransferases from acetogenic and methanogenic organisms, benzimidazole is dissociated from cobalt, but without replacement by histidine. Thus, dimethylbenzimidazole displacement appears to be an emerging theme in cobamide-containing methyltransferases. In methionine synthase, the best studied of the methyltransferases, the histidine ligand appears to be required for competent methyl transfer between methyl- tetrahydrofolate and homoc...

Structure-Based Perspectives on B12-Dependent Enzymes

Phthalate dioxygenase reductase (PDR) is a prototypical iron-sulfur flavoprotein (36 kilodaltons) that utilizes flavin mononucleotide (FMN) to mediate electron transfer from the two-electron donor, reduced nicotinamide adenine nucleotide (NADH), to the one-electron acceptor, [2Fe-2S]. The crystal structure of oxidized PDR from Pseudomonas cepacia has been analyzed at 2.0 angstrom resolution resolution; reduced PDR and pyridine nucleotide complexes have been analyzed at 2.7 angstrom resolution. NADH, FMN, and the [2Fe-2S] cluster, bound to distinct domains, are brought together near a central cleft in the molecule, with only 4.9 angstroms separating the flavin 8-methyl and a cysteine sulfur ligated to iron. The domains that bind FMN and [2Fe-2S] are packed so that the flavin ring and the plane of the [2Fe-2S] core are approximately perpendicular. The [2Fe-2S] group is bound by four cysteines in a site resembling that in plant ferredoxins, but its redox potential (-174 millivolts at pH 7.0) is much higher than the potentials of plant ferredoxins. Structural and sequence similarities assign PDR to a distinct family of flavoprotein reductases, all related to ferredoxin NADP(+)-reductase.

https://science.sciencemag.org/content/sci/258/5088/1604.full.pdf

Phthalate Dioxygenase Reductase: A Modular Structure for Electron Transfer from Pyridine Nucleotides to [2Fe-2S]

The crystal structure of dimeric Fe(II1) superoxide dismutase (SOD) from Escherichia coli (3006 protein atoms, 2 irons, and 28 1 solvents) has been refined to an R of 0.184 using all observed data between 40.0 and 1.85 8, (34 879 reflections). Features of this structure are compared with the refined structure of MnSOD from Thermus thermophilus. The coordination geometry at the Fe site is distorted trigonal bipyramidal, with axial ligands His26 and solvent (proposed to be OH-), and in-plane ligands His73, Asp156, and Hisl6O. Reduction of crystals to the Fe(I1) state does not result in significant changes in metal-ligand geometry (R = 0.188 for data between 40.0 and 1.80 A). The arrangement of iron ligands in Fe(I1) and Fe(II1)SOD closely matches the Mn coordination found in MnSOD from T. thermophilus (Ludwig, M. L., Metzger, A. L., Pattridge, K. A., & Stallings, W. C. (1991) J. Mol. Biol. 219, 335-3581. Structures of the Fe(II1) azide (40.0-1.8 A, R = 0.186) and Mn(II1) azide (20.0-1.8 A, R = 0.179) complexes, reported here, reveal azide bound as a sixth ligand with distorted octahedral geometry at the metal; the in-plane ligand-Fe-ligand and ligand-Mn-ligand angles change by 20-30" to coordinate azide as a sixth ligand. However, the positions of the distal azide nitrogens are different in the FeSOD and MnSOD complexes. The geometries of the Fe(III), Fe(II), and Fe(III)-azide species suggest a reaction mechanism for superoxide dismutation in which the metal alternates between five- and six-coordination. A reaction scheme in which the ligated solvent acts as a proton acceptor in the first half-reaction (formation of Fe(I1) and oxygen) is consistent with the pH dependence of the kinetic parameters and spectroscopic properties of Fe superoxide dismutase.

Martha L. Ludwig

Papers

The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.

Thioredoxin reductase two modes of catalysis have evolved.

Structure-Based Perspectives on B12-Dependent Enzymes

Phthalate Dioxygenase Reductase: A Modular Structure for Electron Transfer from Pyridine Nucleotides to [2Fe-2S]

Structure-function in Escherichia coli iron superoxide dismutase: comparisons with the manganese enzyme from Thermus thermophilus.