Role of potassium levels in pkBADH heterogeneity of NAD+ binding site

Muñoz-Bacasehua, César; Rosas‐Rodríguez, Jesús A.; Arvizu‐Flores, Aldo A.; Valenzuela‐Soto, Elisa M.

doi:10.1007/s10863-020-09827-7

Cited by 5 publications

(7 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These findings indicate that K + ions have a stabilizing effect on pkBADH. In a recent study published by our working group, 26 we demonstrated that in the absence of NAD + , 25 mM potassium increases the thermal stability of pkBADH because it modifies the secondary structure of pkBADH. It is interesting that in the presence of NAD + , potassium is able to relax the enzyme secondary structure to increase its thermal stability.…”

Section: Resultsmentioning

confidence: 73%

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Muñoz-Bacasehua

Rosas‐Rodríguez²,

Arvizu‐Flores

et al. 2020

J of Molecular Recognition

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 73%

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Muñoz-Bacasehua

Rosas‐Rodríguez²,

Arvizu‐Flores

et al. 2020

J of Molecular Recognition

View full text Add to dashboard Cite

show abstract

“…The binding of K + to the enzyme has been proposed for pkBADH and other BADHs, where K + can bind to different sites: (1) amino acid residues located in the coenzyme binding domain (called intramolecular subunit cavity), (2) in the intersubunit cavity, and (3) in the central cavity of the tetramer. [10,12,13] The binding of K + into previously described pkBADH sites, could cause residue conformational changes or modification of the tertiary structure. Garza-Ramos et al found a similar effect in PaBADH, where changes in the protein's tertiary structure were dependent on the K + concentration.…”

Section: Variable Potassium Concentrationsmentioning

confidence: 99%

“…The analysis showed n approximates to 1, suggesting a unique type of site with the same ous studies that demonstrated that K + modified the affinity of the enzyme for the NAD + . [10] 3.3 | Effect of K + on secondary structure content of pkBADH CD spectra of the pkBADH-NAD + complex in the absence of K + exhibited the characteristics of electronic transitions of negative absorption bands with maxima at $208 nm (n!π*) and 222 nm (π!π*), which are the characteristic bands of the α-helical structure of the pkBADH-NAD + complex (Figure 6a). The CD data obtained from the pkBADH-NAD + complex in the absence of K showed different ellipticity values to the enzyme with NAD + at each concentration of KCl evaluated (Figure 6a).…”

Section: Binding Sites On Pkbadhmentioning

confidence: 99%

“…[9] A recent study published by our group found that NAD + binding to the first active site of pkBADH decreased the affinity of the coenzyme for the second active site, whereas the potassium presence increased the affinity to each active site. [10] Similarly, NAD + binding to pkBADH provoked changes in the pkBADH secondary structure content and thermal stability; however, those changes were influenced by potassium. [11] The potassium binding sites have been described in PaBADH, LvBADH, and pkBADH previously; all the enzymes share the intracavity and the intercavity binding sites, while the central cavity has not been found for pkBADH.…”

Section: Introductionmentioning

confidence: 99%

“…[11] The potassium binding sites have been described in PaBADH, LvBADH, and pkBADH previously; all the enzymes share the intracavity and the intercavity binding sites, while the central cavity has not been found for pkBADH. [10,12,13] The concentration of potassium ions changes in the cytoplasm of renal medulla cells depending on whether it is in the form of concentrated urine or dilute urine. During the formation of concentrated urine, synthesis and accumulation of GB are required, and synthesized by pkBADH.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase

et al. 2021

View full text Add to dashboard Cite

Role of potassium levels in pkBADH heterogeneity of NAD+ binding site

Cited by 5 publications

References 36 publications

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase

Contact Info

Product

Resources

About