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Carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis.

ABSTRACT: 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.

SUBMITTER: Zachos I

PROVIDER: S-EPMC8252718 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis.

Zachos Ioannis I Döring Manuel M Tafertshofer Georg G Simon Robert C RC Sieber Volker V

Angewandte Chemie (International ed. in English) 20210510 26

Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba-NADP<sup>+</sup> ) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP<sup>+</sup> 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 ...[more]

PMID: 33719153

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Dataset Information

Carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis.

Publications

carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis.

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