- Chlamydia trachomatis glyceraldehyde 3-phosphate dehydrogenase: Enzyme kinetics, high-resolution crystal structure, and plasminogen binding
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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular ‘moonlighting’ functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism.
- Schormann, Norbert,Campos, Juan,Motamed, Rachael,Hayden, Katherine L.,Gould, Joseph R.,Green, Todd J.,Senkovich, Olga,Banerjee, Surajit,Ulett, Glen C.,Chattopadhyay, Debasish
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- carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis
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Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba-NADP+) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half-life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium-N?glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba-NADP+ opens up the field of redox-biocatalysis under harsh conditions.
- D?ring, Manuel,Sieber, Volker,Simon, Robert C.,Tafertshofer, Georg,Zachos, Ioannis
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supporting information
p. 14701 - 14706
(2021/05/13)
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- Oxidase-mimicking activity of ultrathin MnO2 nanosheets in a colorimetric assay of chlorothalonil in food samples
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Chlorothalonil is a class of 2B carcinogen which is widely used in the prevention and treatment of fungal diseases in food samples. Its residual problem has been increasingly concerned by society. In this paper, a fast and simple colorimetric assay based on Manganese dioxide nanosheets (MnO2 NSs)-oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) platform was used to detect residual pesticide chlorothalonil in food samples. Under optimal conditions, the half maximal inhibitory concentration and the limit of detection of chlorothalonil were 3.27 and 0.024 ng/mL. There were no obvious cross-reactivity between chlorothalonil and interference substances. The recoveries shown the satisfactory results. The results of colorimetric assay for the authentic samples were largely consistent with gas chromatography. Therefore, the proposed method would be convenient and satisfactory analytical methods for the monitoring of chlorothalonil. Furthermore, the MnO2 – TMB system was used to produce test strips for quick and convenient visual detection of chlorothalonil with good performance.
- Dai, Zhihui,Li, Zhenxi,Lu, Yuxiao,Sheng, Enze,Tan, Yuting,Xiao, Yue
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- Kinetic and mechanistic characterization of the glyceraldehyde 3-phosphate dehydrogenase from Mycobacterium tuberculosis
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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic protein responsible for the conversion of glyceraldehyde 3-phosphate (G3P), inorganic phosphate and nicotinamide adenine dinucleotide (NAD+) to 1,3-bisphosphoglycerate (1,3-BPG) and the reduced form of nicotinamide adenine dinucleotide (NADH). Here we report the characterization of GAPDH from Mycobacterium tuberculosis (Mtb). This enzyme exhibits a kinetic mechanism in which first NAD+, then G3P bind to the active site resulting in the formation of a covalently bound thiohemiacetal intermediate. After oxidation of the thiohemiacetal and subsequent nucleotide exchange (NADH off, NAD+ on), the binding of inorganic phosphate and phosphorolysis yields the product 1,3-BPG. Mutagenesis and iodoacetamide (IAM) inactivation studies reveal the conserved C158 to be responsible for nucleophilic catalysis and that the conserved H185 to act as a catalytic base. Primary, solvent and multiple kinetic isotope effects revealed that the first half-reaction is rate limiting and utilizes a step-wise mechanism for thiohemiacetal oxidation via a transient alkoxide to promote hydride transfer and thioester formation.
- Wolfson-Stofko, Brett,Hadi, Timin,Blanchard, John S.
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- Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs
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Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine l-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of l-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both d- and l-configuration. Human PGK demonstrated catalytic efficiencies in the 104-10 5 M-1 s-1 range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in d- or l-configuration. In contrast, it was poorly active with natural pyrimidine d-nucleotides (less than 103 M-1 s-1). Pyrimidine l-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their d-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, l-2′-deoxycytosine and l-2′-deoxythymidine.
- Gallois-Montbrun, Sarah,Faraj, Abdesslem,Seclaman, Edward,Sommadossi, Jean-Pierre,Deville-Bonne, Dominique,Véron, Michel
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p. 1749 - 1756
(2007/10/03)
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