Insights into the mechanism of type I dehydroquinate dehydratases from structures of reaction intermediates

The biosynthetic shikimate pathway consists of seven enzymes that catalyze sequential reactions to generate chorismate, a critical branch point in the synthesis of the aromatic amino acids. The third enzyme in the pathway, dehydroquinate dehydratase (DHQD), catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate. We present three crystal structures of the type I DHQD from the intestinal pathogens Clostridium difficile and Salmonella enterica. Structures of the enzyme with substrate and covalent pre- and post-dehydration reaction intermediates provide snapshots of successive steps along the type I DHQD-catalyzed reaction coordinate. These structures reveal that the position of the substrate within the active site does not appreciably change upon Schiff base formation. The intermediate state structures reveal a reaction state-dependent behavior of His-143 in which the residue adopts a conformation proximal to the site of catalytic dehydration only when the leaving group is present. We speculate that His-143 is likely to assume differing catalytic roles in each of its observed conformations. One conformation of His-143 positions the residue for the formation/hydrolysis of the covalent Schiff base intermediates, whereas the other conformation positions the residue for a role in the catalytic dehydration event. The fact that the shikimate pathway is absent from humans makes the enzymes of the pathway potential targets for the development of non-toxic antimicrobials. The structures and mechanistic insight presented here may inform the design of type I DHQD enzyme inhibitors.     

Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues.

In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography. Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79. In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79. The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor. The mode of interaction of His79 is, however, different. In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues. In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues). These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79. The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase.     

Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.     

Structural analysis of Golgi alpha-mannosidase II inhibitors identified from a focused glycosidase inhibitor screen

The N-glycosylation pathway is a target for pharmaceutical intervention in a number of pathological conditions including cancer. Golgi alpha-mannosidase II (GMII) is the final glycoside hydrolase in the pathway and has been the target for a number of synthetic efforts aimed at providing more selective and effective inhibitors. Drosophila GMII (dGMII) has been extensively studied due to the ease of obtaining high resolution structural data, allowing the observation of substrate distortion upon binding and after formation of a trapped covalent reaction intermediate. However, attempts to find new inhibitor leads by high-throughput screening of large commercial libraries or through in silico docking were unsuccessful. In this paper we provide a kinetic and structural analysis of five inhibitors derived from a small glycosidase-focused library. Surprisingly, four of these were known inhibitors of beta-glucosidases. X-ray crystallographic analysis of the dGMII:inhibitor complexes highlights the ability of the zinc-containing GMII active site to deform compounds, even ones designed as conformationally restricted transition-state mimics of beta-glucosidases, into binding entities that have inhibitory activity. Although these deformed conformations do not appear to be on the expected conformational itinerary of the enzyme, and are thus not transition-state mimics of GMII, they allow positioning of the three vicinal hydroxyls of the bound gluco-inhibitors into similar locations to those found with mannose-containing substrates, underlining the importance of these hydrogen bonds for binding. Further, these studies show the utility of targeting the acid-base catalyst using appropriately positioned positively charged nitrogen atoms, as well as the challenges associated with aglycon substitutions.     

Crystal structures of arginine deiminase with covalent reaction intermediates; implications for catalytic mechanism

Arginine deiminase (ADI), an enzyme that hydrolyzes arginine to generate energy in many parasitic microorganisms, has potent anticancer activities and can halt growth of solid tumors. We determined the crystal structure of ADI from Mycoplasma arginini in two different forms (1.6 and 2.0 A resolution) using multiple isomorphous replacement. ADI shares common structural features with the arginine-catabolizing enzymes Arg:Gly amidinotransferase and dimethylarginine dimethyl-aminohydrolase; ADI contains an additional domain of five helices. The scissile C-N bonds of the substrates and the catalytic triads (Cys398-His269-Glu213 of ADI) for the three enzymes superimpose on each other. The ADI structure from form I crystals corresponds to a tetrahedral intermediate with four heteroatoms (1S, 2N, 1O) covalently bonded to the reaction-center carbon. The structure from form II crystals represents an amidino-enzyme complex; the reaction-center carbon is covalently bonded to Cys398 sulfur and two nitrogens, and the reacting water molecule is only 2.54 A away.     

Insights into brain development and disease from neurogenetic analyses in Drosophila melanogaster

Groundbreaking work by Obaid Siddiqi has contributed to the powerful genetic toolkit that is now available for studying the nervous system of Drosophila. Studies carried out in this powerful neurogenetic model system during the last decade now provide insight into the molecular mechanisms that operate in neural stem cells during normal brain development and during abnormal brain tumorigenesis. These studies also provide strong support for the notion that conserved molecular genetic programs act in brain development and disease in insects and mammals including humans.     

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.     

Insights into the structural basis of the pH-dependent dimer-tetramer equilibrium through crystallographic analysis of recombinant Diocleinae lectins

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.     

Comparative structural analysis of oxidized and reduced thioredoxin from Drosophila melanogaster

Thioredoxins (Trx) participate in essential antioxidant and redox-regulatory processes via a pair of conserved cysteine residues. In dipteran insects like Drosophila and Anopheles, which lack a genuine glutathione reductase (GR), thioredoxins fuel the glutathione system with reducing equivalents. Thus, characterizing Trxs from these organisms contributes to our understanding of redox control in GR-free systems and provides information on novel targets for insect control. Cytosolic Trx of Drosophila melanogaster (DmTrx) is the first thioredoxin that was crystallized for X-ray diffraction analysis in the reduced and in the oxidized form. Comparison of the resulting structures shows rearrangements in the active-site regions. Formation of the C32-C35 disulfide bridge leads to a rotation of the side-chain of C32 away from C35 in the reduced form. This is similar to the situation in human Trx and Trx m from spinach chloroplasts but differs from Escherichia coli Trx, where it is C35 that moves upon change of the redox state. In all four crystal forms that were analysed, DmTrx molecules are engaged in a non-covalent dimer interaction. However, as demonstrated by gel-filtration analyses, DmTrx does not dimerize under quasi in vivo conditions and there is no redox control of a putative monomer/dimer equilibrium. The dimer dissociation constants K(d) were found to be 2.2mM for reduced DmTrx and above 10mM for oxidized DmTrx as well as for the protein in the presence of reduced glutathione. In human Trx, oxidative dimerization has been demonstrated in vitro. Therefore, this finding may indicate a difference in redox control of GR-free and GR-containing organisms.     

Insights into protein-DNA interactions through structure network analysis

Protein-DNA interactions are crucial for many cellular processes. Now with the increased availability of structures of protein-DNA complexes, gaining deeper insights into the nature of protein-DNA interactions has become possible. Earlier, investigations have characterized the interface properties by considering pairwise interactions. However, the information communicated along the interfaces is rarely a pairwise phenomenon, and we feel that a global picture can be obtained by considering a protein-DNA complex as a network of noncovalently interacting systems. Furthermore, most of the earlier investigations have been carried out from the protein point of view (protein-centric), and the present network approach aims to combine both the protein-centric and the DNA-centric points of view. Part of the study involves the development of methodology to investigate protein-DNA graphs/networks with the development of key parameters. A network representation provides a holistic view of the interacting surface and has been reported here for the first time. The second part of the study involves the analyses of these graphs in terms of clusters of interacting residues and the identification of highly connected residues (hubs) along the protein-DNA interface. A predominance of deoxyribose-amino acid clusters in beta-sheet proteins, distinction of the interface clusters in helix-turn-helix, and the zipper-type proteins would not have been possible by conventional pairwise interaction analysis. Additionally, we propose a potential classification scheme for a set of protein-DNA complexes on the basis of the protein-DNA interface clusters. This provides a general idea of how the proteins interact with the different components of DNA in different complexes. Thus, we believe that the present graph-based method provides a deeper insight into the analysis of the protein-DNA recognition mechanisms by throwing more light on the nature and the specificity of these interactions.     

Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.     

Insights from the reconstitution of the divergent outer kinetochore of Drosophila melanogaster

Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.     

The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure

Summary Golgi studies of the neurons in the optic lobes of Drosophila melanogaster reveal a large number of neuronal cell types. These can be classified as either columnar or tangential. Columnar elements establish the retinotopic maps of the lamina, medulla, and lobula-complex neuropiles. They are classified according to the position of their cell bodies, the number, width, and level of their arborizations, and their projection areas. Tangential elements are oriented perpendicularly to the columns. The arborizations of different tangential neurons are restricted to different layers of the optic neuropiles, within such layers their dendritic fields may span the entire retinotopic field or only part of it. The abundance of cell types inside each of the columnar units of the optic lobe is discussed with regard to its possible functional significance. By means of their stratified arborizations the columnar neurons form what appear to be multiple sets of retinotopically organized parallel information processing networks. It is suggested that these parallel networks filter different kinds of visual information and thus represent structurally separated functional subunits of the optic lobe. Such a parallel organization of visual functions increases the sites for function-specific gene actions and may explain the behavioral phenotypes of recently isolated structural mutants of the optic lobe.     

Insights into the mechanism of prion propagation

Proteins with prion properties have been identified in both mammals and fungi. The tractability of yeast as a genetic model has contributed significantly to our understanding of prion formation and propagation. A number of molecular chaperones have been found to modulate the ability of yeast prion proteins to propagate. The results of recent genetic and in vitro studies have shed light on the mechanism of prion propagation, the physical and structural basis of different prion strains and the species barrier, as well as the function and mechanism of the chaperones that interact with the prion proteins. Whether aspects of the mechanisms of formation, maintenance and clearance of prions are conserved between fungi and mammals remains to be seen.     

NMR analysis of covalent intermediates in thiamin diphosphate enzymes

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.