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Abstract
The enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) plays a central role in the nicotinamide and adenine dinucleotide (NAD) synthesis as its de novo and salvage pathways converge in it. NAD is a crucial molecule in the metabolism of all living beings, mainly in redox metabolism. Here we describe a new methodological strategy for the evaluation of possible inhibitors of Leishmania braziliensis NMNAT (LbNMNAT). Our method suppresses the cross inhibitory activity of the enzyme alcohol dehydrogenase (ADH, EC 1.1.1.1) coupled to the detection assay. We improved the specificity of the coupled enzymatic system, by introducing an additional solid-phase extraction step of the inhibitors prior to the execution of the coupled detection system. This step enabled the differentiated evaluation of the LbNMNAT activity without interfering in the coupled ADH enzyme, as well as the assessment of the inhibitory activity of gallotannin, an inhibitor found in Rhus chinensis, on LbNMNAT.
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