(172d) Aberrant NADH Molecules: Toxicity and Remediation

Oxidoreductases rely on a limited pool of coenzymes, such as NAD⁺/NADH, to catalyze redox reactions. However, in biological systems, aberrant isomers of NADH, such as 1,2-NADH and 1,6-NADH, can spontaneously generate in addition to the biologically active 1,4-NADH. These aberrant isomers cannot be used by oxidoreductases, leading to depletion of the cofactor pool and inhibition of dehydrogenases. Therefore, the recovery of these aberrant isomers into usable forms is essential for increasing the efficiency of continuous processes.

In this study, a concentrated solution of NAD+ (34 mM) and 1,4-NADH (1.5 M) was prepared, and a rate of 5 μM h^-1 of spontaneous 1,6-NADH was recorded. The accumulation of these isomers will also be monitored in dilute solutions and physiological systems. The inhibition of central metabolism and individual enzymes using purified 1,6-NADH will also be investigated. Succinic acid (SA) production will be used as the model process to analyze its titers, serving as an indirect measure of the inhibition of its biosynthetic pathway by the aberrant molecule. Finally, enzyme-mediated recovery will be employed to mitigate their formation and increase the efficacy of the process. Mutant human renalase, generated previously in our group, showed enhanced catalytic activity in preliminary experiments, and could be used in mitigating the toxic effects of these aberrant isomers by oxidizing them into NAD⁺.

Studying the generation of these isomers and their subsequent detoxification will enhance the efficiency of cofactor regeneration and, consequently, in vitro biosynthetic pathways.