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Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi‐bi ordered mechanism in which NAD+ binds to the enzyme before the aldehyde. Previous studies showed that NAD+ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD+ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD+ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD+. The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD+ to the enzyme caused changes in its secondary structure, whereas the presence of K+ helps maintain its α‐helix content. K+ increased the thermal stability of the pkBADH‐NAD+ complex by 5.3°C. ITC data showed that NAD+ binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD+ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.

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Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD + as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD + with different affinities at each active site and that the binding is K + dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD + and the role played by K + . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K + and titrating the enzyme with varying concentrations of NAD + . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K + was increased. Changes in the exposure of tryptophan located near the NAD + binding site were found when the enzyme was titrated with NAD + in the presence of potassium. Fluorescence data analysis showed that the K + presence promoted static quenching that facilitated the pkBADH-NAD + complex formation. DC data analysis showed that binding of K + to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K + , respectively. The presence of K + during NAD + binding to pkBADH increased the thermal stability of the complex. These results indicated that K + facilitated the pkBADH-NAD + complex formation and suggested that K + caused small changes in secondary and tertiary structures that could influence the active site conformation.