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2.
FEBS Open Bio ; 5: 292-302, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25905034

RESUMO

Eukaryotic aminopeptidase P1 (APP1), also known as X-prolyl aminopeptidase (XPNPEP1) in human tissues, is a cytosolic exopeptidase that preferentially removes amino acids from the N-terminus of peptides possessing a penultimate N-terminal proline residue. The enzyme has an important role in the catabolism of proline containing peptides since peptide bonds adjacent to the imino acid proline are resistant to cleavage by most peptidases. We show that recombinant and catalytically active Caenorhabditis elegans APP-1 is a dimer that uses dinuclear zinc at the active site and, for the first time, we provide structural information for a eukaryotic APP-1 in complex with the inhibitor, apstatin. Our analysis reveals that C. elegans APP-1 shares similar mode of substrate binding and a common catalytic mechanism with other known X-prolyl aminopeptidases.

3.
J Med Chem ; 55(12): 5841-50, 2012 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-22621375

RESUMO

The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.


Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Malária/parasitologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Plasmodium falciparum/enzimologia , Di-Hidro-Orotato Desidrogenase , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Conformação Proteica , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato
4.
J Neural Transm (Vienna) ; 118(7): 1043-53, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21547391

RESUMO

Copper amine oxidases are important for the metabolism of a range of biogenic amines. Here, we focus on substrate specificity in the E. coli copper amine oxidase (ECAO) and specifically the role of Tyr 381. This residue, and its equivalent, in other copper amine oxidases has been referred to as a "gating" residue able to move position depending upon the presence or absence of amine substrate. The position of this residue suggests a role in substrate selectivity. We have compared the properties of two variant forms of ECAO, Y381F and Y381A, with wild-type enzyme by steady-state kinetics of oxidation of a number of amine substrates, modes of inhibitor interactions and X-ray structure determination. Y381F displays a similar catalytic efficiency to wild type against the preferred substrate ß-phenylethylamine. In both cases oxidation of the alternative aromatic amine substrate benzylamine is relatively poor, although Y381F represents an efficient benzylamine oxidase. By contrast, Y381A performed poorly against both aromatic substrates predominantly due to an increased K (M) which we propose is due to the lack of an aromatic residue to orient substrate towards the TPQ and active site base. These results are supported by different behaviour of Y381A to inhibition with 2-hydrazinopyridine. We also report on methylamine turnover by the three enzymes. We propose that Y381, together with another residue Y387, may be considered of critical importance for the substrate selectivity of ECAO, through stacking or hydrophobic interactions with substrate.


Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/fisiologia , Escherichia coli/enzimologia , Tirosina/química , Tirosina/fisiologia , Amina Oxidase (contendo Cobre)/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiologia , Metilaminas/química , Piridonas/química , Especificidade por Substrato/genética , Tirosina/genética
5.
J Med Chem ; 52(9): 2683-93, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19351152

RESUMO

Pyrimidine biosynthesis is an attractive drug target in a variety of organisms, including humans and the malaria parasite Plasmodium falciparum. Dihydroorotate dehydrogenase, an enzyme catalyzing the only redox reaction of the pyrimidine biosynthesis pathway, is a well-characterized target for chemotherapeutical intervention. In this study, we have applied SPROUT-LeadOpt, a software package for structure-based drug discovery and lead optimization, to improve the binding of the active metabolite of the anti-inflammatory drug leflunomide to the target cavities of the P. falciparum and human dihydroorotate dehydrogenases. Following synthesis of a library of compounds based upon the SPROUT-optimized molecular scaffolds, a series of inhibitors generally showing good inhibitory activity was obtained, in keeping with the SPROUT-LeadOpt predictions. Furthermore, cocrystal structures of five of these SPROUT-designed inhibitors bound in the ubiquinone binding cavity of the human dihydroorotate dehydrogenase are also analyzed.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo
7.
J Biol Chem ; 277(36): 33115-26, 2002 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-12065585

RESUMO

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.


Assuntos
Chaperonina 60/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Íons , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Temperatura
8.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 3): 421-30, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11856827

RESUMO

In the Gram-positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled by the transcriptional regulator AhrC. The hexameric AhrC protein binds in an L-arginine-dependent manner to pseudo-palindromic operators within the promoter regions of arginine biosynthetic and catabolic gene clusters. AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription of catabolic genes. The crystal structure of AhrC has been determined at 2.7 A resolution. Each subunit of the protein has two domains. The C-terminal domains are arranged with 32 point-group symmetry and mediate the major intersubunit interactions. The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not obey strict symmetry. A structural comparison of AhrC with the arginine repressor from the thermophile B. stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition, but also reveals some striking sequence differences, especially within the C-terminal oligomerization domain, which may contribute to the different thermostabilities of the proteins. Comparison of the crystal structure of AhrC with a 30 A resolution model obtained by combining X-ray structure-factor amplitudes with phases derived from electron-microscopic analyses of AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more widely useful for obtaining low-resolution phase information.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas Repressoras/química , Transativadores/química , Sequência de Aminoácidos , Biopolímeros/química , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
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