RESUMO
Glyoxalase system is an ubiquitous system in human cells which has been examined thoroughly for its role in different diseases. It comprises two enzymes; Glyoxalase I (Glo-I) and Glyoxalase II (Glo-II) which perform detoxifying endogenous harmful metabolites, mainly methylglyoxal (MG) into non-toxic bystanders. In silico computer Aided Drug Design approaches were used and ninety two diverse pharmacophore models were generated from eighteen Glyoxalase I crystallographic complexes. Subsequent QSAR modeling followed by ROC evaluation identified a single pharmacophore model which was able to predict the expected Glyoxalase I inhibition. Screening of the National Cancer Institute (NCI) database using the optimal pharmacophore Hypo(3VW9) identified several promising hits. Thirty eight hits were successfully predicted then ordered and evaluated in vitro. Seven hits out of the thirty eight tested compounds showed more than 50% inhibition with low micromolar IC50.
Assuntos
Antineoplásicos/metabolismo , Inibidores Enzimáticos/metabolismo , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/metabolismo , Antineoplásicos/química , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Lactoilglutationa Liase/química , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Curva ROC , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
Glyoxalase I is the first enzyme of the glyoxalase system that can detoxify methylglyoxal, a cytotoxic compound increased rapidly under stress conditions. Here we report cloning and characterization of a glyoxalase I from sugar beet M14 line (an interspecific hybrid between a wild species Beta corolliflora Zoss and a cultivated species B. vulgaris L). The full-length gene BvM14-glyoxalase I has 1,449 bp in length with an open reading frame of 1,065 bp encoding 354 amino acids. Sequence analysis shows the conserved glyoxalase I domains, metal and glutathione binding sites and secondary structure (α-helixes and ß-sheets). The BvM14-glyoxalase I gene was ubiquitously expressed in different tissues of sugar beet M14 line and up-regulated in response to salt, mannitol and oxidative stresses. Heterologous expression of BvM14-glyoxalase I could increase E. coli tolerance to methylglyoxal. Transgenic tobacco plants constitutively expressing BvM14-glyoxalase I were generated. Both leaf discs and seedlings showed significant tolerance to methylglyoxal, salt, mannitol and H2O2. These results suggest an important role of BvM14-glyoxalase I in cellular detoxification and tolerance to abiotic stresses.
Assuntos
Beta vulgaris/genética , Lactoilglutationa Liase/genética , Nicotiana/genética , Proteínas de Plantas/genética , Aldeído Pirúvico/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Beta vulgaris/enzimologia , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Lactoilglutationa Liase/química , Lactoilglutationa Liase/metabolismo , Manitol/metabolismo , Dados de Sequência Molecular , Estresse Oxidativo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Cloreto de Sódio/metabolismo , Nicotiana/metabolismoRESUMO
Glyoxalase I was highly purified from onion bulbs by DEAE-cellulose, hydroxyapatite, and S-hexylglutathione-agarose column chromatography. With 356 micromol min(-1) mg(-1) protein, the specific enzymatic activity of the purified enzyme is the highest reported to date in plants. The purified enzyme showed a single major band with a relative molecular mass of approximately 25,000 on SDS-PAGE. A cDNA encoding glyoxalase I was cloned and sequenced. Sequence comparison suggested that it is to be classified as a short-type glyoxalase I. The expression pattern of glyoxalase I in healthy onion plants and responses to various stresses were examined by Western blotting. Glyoxalase I exists at high concentration in roots, young bulbs, mature bulbs, and mature leaves, the highest concentration being in mature bulbs. Up-regulation of glyoxalase I and glyoxalase II enzyme activities were observed in response to various stresses, and an increase in Gly I protein was also seen by immunoblotting. Our results suggest an important role of the glyoxalase I gene in the plant abiotic stress response.
Assuntos
Lactoilglutationa Liase/isolamento & purificação , Cebolas/enzimologia , Sequência de Aminoácidos , Western Blotting , Cromatografia Líquida/métodos , Clonagem Molecular , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Lactoilglutationa Liase/química , Lactoilglutationa Liase/genética , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.