Effects of Mutations on the Molecular Dynamics of Oxygen Escape from the Dimeric Hemoglobin of Scapharca inaequivalvis

Like many hemoglobins, the structure of the dimeric hemoglobin from the clam Scapharca inaequivalvis is a “closed bottle” since there is no direct tunnel from the oxygen binding site on the heme to the solvent. The proximal histidine faces the dimer interface, which consists of the E and F helicies. This is significantly different from tetrameric vertebrate hemoglobins and brings the heme groups near the subunit interface. The subunit interface is also characterized by an immobile, hydrogen-bonded network of water molecules. Although there is data which is consistent with the histidine gate pathway for ligand escape, these aspects of the structure would seem to make that pathway less likely. Locally enhanced sampling molecular dynamics are used here to suggest alternative pathways in the wild-type and six mutant proteins. In most cases the point mutations change the selection of exit routes observed in the simulations. Exit via the histidine gate is rarely seem although oxygen molecules do occasionally cross over the interface from one subunit to the other. The results suggest that changes in flexibility and, in some cases, creation of new cavities can explain the effects of the mutations on ligand exit paths

The Dimerization Domain in DapE Enzymes Is Required for Catalysis

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,Ldiaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate

Practical Spectrophotometric Assay for the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics

Mapping of a Regulatory Site of the Escherichia coli ADP-Glucose Pyrophosphorylase

The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) controls the biosynthesis of glycogen in bacteria and starch in plants. It is regulated by various activators in different organisms according to their metabolic characteristics. In Escherichia coli, the major allosteric activator is fructose 1,6-bisphosphate (FBP). Other potent activator analogs include 1,6-hexanediol bisphosphate (HBP) and pyridoxal 5′-phosphate (PLP). Recently, a crystal structure with FBP bound was reported (PDB ID: 5L6S). However, it is possible that the FBP site found is not directly responsible for the activation of the enzyme. We hypothesized FBP activates by binding one of its phosphate groups to another site (“P1”) in which a sulfate molecule was observed. In the E. coli enzyme, Arg40, Arg52, and Arg386 are part of this “P1” pocket and tightly complex this sulfate, which is also present in the crystal structures of ADP-Glc PPases from Agrobacterium tumefaciens and Solanum tuberosum. To test this hypothesis, we modeled alternative binding conformations of FBP, HBP, and PLP into “P1.” In addition, we performed a scanning mutagenesis of Arg residues near potential phosphate binding sites (“P1,” “P2,” “P3”). We found that Arg40 and Arg52 are essential for FBP and PLP binding and activation. In addition, mutation of Arg386 to Ala decreased the apparent affinity for the activators more than 35-fold. We propose that the activator binds at this “P1” pocket, as well as “P2.” Arg40 and Arg52 are highly conserved residues and they may be a common feature to complex the phosphate moiety of different sugar phosphate activators in the ADP-Glc PPase family

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism toward the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme toward the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near ~600-fold higher that other nucleotides, whereas in the absence of activator was only ~3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. University of Chicago; Estados UnidosFil: Solamen, Ligin. University of Chicago; Estados UnidosFil: Hill, Benjamin L.. University of Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Olsen, Kenneth W.. University of Chicago; Estados UnidosFil: Ballicora, Miguel A.. University of Chicago; Estados Unido

Unraveling the Activation Mechanism of the Potato Tuber ADP-glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina;Fil: Kuhn, Misty L.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Falaschetti, Christine A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Solamen, Ligin. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Olsen, Kenneth W.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Ballicora, Miguel A.. Loyola University. Dept. of Chemistry and Biochem.; Estados Unidos de América;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

Regulation of Gonad Morphogenesis in Drosophila melanogaster by BTB Family Transcription Factors

During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. We have characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-à-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, we demonstrate that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, we find that Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis

Low-dose arsenic compromises the immune response to influenza A infection in vivo

This paper is not subject to U.S. copyright. The definitive version was published in Environmental Health Perspectives 117 (2009): 1441–1447, doi:10.1289/ehp.0900911.Arsenic exposure is a significant worldwide environmental health concern. We recently reported that 5-week exposure to environmentally relevant levels (10 and 100 ppb) of As in drinking water significantly altered components of the innate immune response in mouse lung, which we hypothesize is an important contributor to the increased risk of lung disease in exposed human populations. We investigated the effects of As exposure on respiratory influenza A (H1N1) virus infection, a common and potentially fatal disease. In this study, we exposed C57BL/6J mice to 100 ppb As in drinking water for 5 weeks, followed by intranasal inoculation with a sublethal dose of influenza A/PuertoRico/8/34 (H1N1) virus. Multiple end points were assessed postinfection. Arsenic was associated with a number of significant changes in response to influenza, including an increase in morbidity and higher pulmonary influenza virus titers on day 7 postinfection. We also found many alterations in the immune response relative to As-unexposed controls, including a decrease in the number of dendritic cells in the mediastinal lymph nodes early in the course of infection. Our data indicate that chronic As exposure significantly compromises the immune response to infection. Alterations in response to repeated lung infection may also contribute to other chronic illnesses, such as bronchiectasis, which is elevated by As exposure in epidemiology studies.This work was supported by grant P42 ES007373 from the National Institute of Environmental Health Sciences [J.W.H.; Superfund Basic Research Program (SBRP) Project 2]. C.D.K. was supported by a graduate fellowship from P42 ES007373 (SBRP, Training Core) and by National Institutes of Health predoctoral fellowship T32-DF007301