scholarly journals Preliminary Characterization of a Ni2+-Activated and Mycothiol-Dependent Glyoxalase I Enzyme from Streptomyces coelicolor

Inorganics ◽  
2019 ◽  
Vol 7 (8) ◽  
pp. 99 ◽  
Author(s):  
Uthaiwan Suttisansanee ◽  
John F. Honek
Keyword(s):  
Leishmania Major ◽  
Homo Sapiens ◽  
Streptomyces Coelicolor ◽  
Glyoxalase I ◽  
Glyoxalase System ◽  
Genetic Studies ◽  
Preliminary Characterization ◽  
Glyoxalase Ii ◽  
Activation Profile

The glyoxalase system consists of two enzymes, glyoxalase I (Glo1) and glyoxalase II (Glo2), and converts a hemithioacetal substrate formed between a cytotoxic alpha-ketoaldehyde, such as methylglyoxal (MG), and an intracellular thiol, such as glutathione, to a non-toxic alpha-hydroxy acid, such as d-lactate, and the regenerated thiol. Two classes of Glo1 have been identified. The first is a Zn2+-activated class and is exemplified by the Homo sapiens Glo1. The second class is a Ni2+-activated enzyme and is exemplified by the Escherichia coli Glo1. Glutathione is the intracellular thiol employed by Glo1 from both these sources. However, many organisms employ other intracellular thiols. These include trypanothione, bacillithiol, and mycothiol. The trypanothione-dependent Glo1 from Leishmania major has been shown to be Ni2+-activated. Genetic studies on Bacillus subtilis and Corynebacterium glutamicum focused on MG resistance have indicated the likely existence of Glo1 enzymes employing bacillithiol or mycothiol respectively, although no protein characterizations have been reported. The current investigation provides a preliminary characterization of an isolated mycothiol-dependent Glo1 from Streptomyces coelicolor. The enzyme has been determined to display a Ni2+-activation profile and indicates that Ni2+-activated Glo1 are indeed widespread in nature regardless of the intracellular thiol employed by an organism.

scholarly journals Trypanothione-dependent glyoxalase I in Trypanosoma cruzi

2006 ◽  
Vol 400 (2) ◽  
pp. 217-223 ◽  
Author(s):  
Neil Greig ◽  
Susan Wyllie ◽  
Tim J. Vickers ◽  
Alan H. Fairlamb
Keyword(s):  
Trypanosoma Cruzi ◽  
Leishmania Major ◽  
Subcellular Fractionation ◽  
Competitive Inhibitor ◽  
Rational Drug Design ◽  
Glyoxalase I ◽  
Glyoxalase System ◽  
Glyoxalase Ii ◽  
Rational Drug ◽  
Metal Cofactor

The glyoxalase system, comprizing glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes, such as methylglyoxal, using glutathione as a cofactor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a cofactor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterization of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal cofactor. In contrast with the L. major enzyme, T. cruzi glyoxalase I was far less fast-idious in its choice of metal cofactor efficiently utilizing cobalt, manganese and zinc. T. cruzi glyoxalase I isomerized hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2–3-fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerized and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a Ki of 5.4±0.6 μM. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in the present study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.

Characterization of the glyoxalase I gene from the vascular wilt fungus Verticillium dahliae

2006 ◽  
Vol 52 (9) ◽  
pp. 816-822 ◽  
Author(s):  
A Klimes ◽  
M J Neumann ◽  
S J Grant ◽  
K F Dobinson
Keyword(s):  
Verticillium Dahliae ◽  
Glyoxalase I ◽  
Vascular Wilt ◽  
Glyoxalase System ◽  
Enhanced Sensitivity ◽  
Acid Protein ◽  
Gene Homologue ◽  
I Gene ◽  
Amino Acid Protein

A glyoxalase I gene homologue (VdGLO1) was identified in the vascular wilt fungus Verticillium dahliae by sequence tag analysis of genes expressed during resting structure development. The results of the current study show that the gene encodes a putative 345 amino acid protein with high similarity to glyoxalase I, which produces S-D-lactoylglutathione from the toxic metabolic by-product methylglyoxal (MG). Disruption of the V. dahliae gene by Agrobacterium tumefaciens-mediated transformation resulted in enhanced sensitivity to MG. Mycelial growth of disruption mutants was severely reduced in the presence of 5 mmol/L MG. In contrast, spore production in liquid medium was abolished at 1 mmol/L MG, although not at physiologically relevant concentrations of ≤100 µmol/L. In this first report on the characterization of a glyoxalase I gene in a vascular wilt pathogen, we found that disruption of VdGLO1 had no discernable effect on the pathogenicity of V. dahliae. These data suggest that while the glyoxalase system is necessary for effectively dealing with catastrophic levels of MG, under normal conditions of growth and infection, other MG detoxification pathways in V. dahliae are able to compensate for the absence of the glyoxalase system.Key words: verticillium wilt, glycolytic methylglyoxal pathway, 2-oxoaldehydes.

scholarly journals Spatial control of gene expression in flies using bacterially derived binary transactivation systems

2020 ◽  
Author(s):  
Stephanie Gamez ◽  
Luis C. Vesga ◽  
Stelia C. Mendez-Sanchez ◽  
Omar S. Akbari
Keyword(s):  
Gene Expression ◽  
Pseudomonas Putida ◽  
Caulobacter Crescentus ◽  
Streptomyces Coelicolor ◽  
Molecular Genetic ◽  
Genetic Studies ◽  
Control Of Gene Expression ◽  
Multiple Systems

AbstractControlling gene expression is an instrumental tool for biotechnology, as it enables the dissection of gene function, affording precise spatial-temporal resolution. To generate this control, binary transactivational systems have been used employing a modular activator consisting of a DNA binding domain(s) fused to activation domain(s). For fly genetics, many binary transactivational systems have been exploited in vivo; however as the study of complex problems often requires multiple systems that can be used in parallel, there is a need to identify additional bipartite genetic systems. To expand this molecular genetic toolbox, we tested multiple bacterially-derived binary transactivational systems in Drosophila melanogaster including the p-CymR operon from Pseudomonas putida, PipR operon from Streptomyces coelicolor, TtgR operon from Pseudomonas putida, and the VanR operon from Caulobacter crescentus. Our work provides the first characterization of these systems in an animal model in vivo. For each system we demonstrate robust tissue-specific spatial transactivation of reporter gene expression, enabling future studies to exploit these transactivational systems for molecular genetic studies.

Glyoxalase Centennial conference: introduction, history of research on the glyoxalase system and future prospects

2014 ◽  
Vol 42 (2) ◽  
pp. 413-418 ◽  
Author(s):  
Naila Rabbani ◽  
Paul J. Thornalley
Keyword(s):  
Physiological Function ◽  
Physiological Stress ◽  
Glyoxalase I ◽  
Future Prospects ◽  
Glyoxalase System ◽  
Introduction History ◽  
Glyoxalase Ii ◽  
History Of Research ◽  
History Of ◽  
The University

On 27–29 November 2013, researchers gathered at the University of Warwick, Coventry, U.K., to celebrate the centennial of the discovery of the glyoxalase pathway. The glyoxalase system was discovered and reported in papers by Carl Neuberg and by Henry Drysdale Dakin and Harold Ward Dudley in 1913. All three were leading extraordinary investigators in the pioneering years of biochemistry. Neuberg proposed glyoxalase as the pathway of mainstream glycolysis and Gustav Embden correctly discounted this, later confirmed by Otto Meyerhof. Albert Szent-Györgyi proposed glyoxalase I as the regulator of cell growth and others discounted this. In the meantime, molecular, structural and mechanistic properties of the enzymatic components of the system, glyoxalase I and glyoxalase II, have been characterized. The physiological function of the glyoxalase pathway of enzymatic defence against dicarbonyl glycation, particularly by endogenous methylglyoxal, now seems secure. We are now in an era of investigation of the regulation of the glyoxalase system where a role in aging and disease, physiological stress and drug resistance and development of healthier foods and new pharmaceuticals is emerging. The history of glyoxalase research illustrates the scientific process of hypothesis proposal, testing and rejection or acceptance with further investigation, standing testament to the need for intuition guided by experience and expertise, as well as indefatigable experimentation.

scholarly journals γδ-Dioxovalerate as a substrate for the glyoxalase enzyme system

1973 ◽  
Vol 135 (4) ◽  
pp. 713-719 ◽  
Author(s):  
Tadeusz Jerzykowski ◽  
Romana Winter ◽  
Wojciech Matuszewski
Keyword(s):  
Enzyme System ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Glyoxalase I ◽  
Glyoxalase System ◽  
Thiol Ester ◽  
Glyoxalase Ii ◽  
Metabolic Functions ◽  
Metabolic Role

1. Crude γδ-dioxovalerate was synthesized from laevulinate by two different methods and was purified by Sephadex chromatography. Some analytical reactions of the compound are described. 2. γδ-Dioxovalerate is a substrate for glyoxalase I and the GSH derivative formed by this enzyme is hydrolysed by glyoxalase II to form d-α-hydroxyglutarate. The Km of glyoxalase I for γδ-dioxovalerate is 1.0×10−3m at pH5.8.3. The u.v.-absorption spectrum of thiol ester, synthesized enzymically from γδ-dioxovalerate and GSH by glyoxalase I, is almost identical with that for S-lactoylglutathione. Some optical properties of this thiol ester were measured. 4. Attempts to show reversibility of the glyoxalase system reactions with d-α-hydroxyglutarate as substrate were unsuccessful. 5. The possible metabolic role of the γδ-dioxovalerate reaction is discussed. It is suggested that one of the metabolic functions of the glyoxalase system may be to provide a mechanism for the entry of this compound into the tricarboxylic acid cycle.

scholarly journals A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

2017 ◽  
Vol 11 ◽  
pp. 117793221771247 ◽  
Author(s):  
Larissa Catharina ◽  
Carlyle Ribeiro Lima ◽  
Alexander Franca ◽  
Ana Carolina Ramos Guimarães ◽  
Marcelo Alves-Ferreira ◽  
...  
Keyword(s):  
Drug Development ◽  
Leishmania Major ◽  
Homo Sapiens ◽  
Protein Structures ◽  
Domain Architecture ◽  
Protein Domain ◽  
Markov Modeling ◽  
Pyruvate Phosphate Dikinase ◽  
Homology Comparison

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development.

Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

1982 ◽  
Vol 47 (02) ◽  
pp. 128-131 ◽  
Author(s):  
F Esnard ◽  
E Dupuy ◽  
A M Dosne ◽  
E Bodevin
Keyword(s):  
Endothelial Cells ◽  
Primary Culture ◽  
Ammonium Sulphate ◽  
Concanavalin A ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Preliminary Characterization ◽  
Umbilical Vein Endothelial Cells ◽  
Ammonium Sulphate Saturation

SummaryA preliminary characterization of a fibrinolytic inhibitor released by human umbilical vein endothelial cells in primary culture is reported. This molecule of Mr comprised between 2 × 105 and 106 and of μ2 mobility precipitates at 43% ammonium sulphate saturation and is totally adsorbed on Concanavalin A Sepharose 4 B. A possible relationship with a macroglobulins is discussed.

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