Potassium and ionic strength effects on the conformational and thermal stability of two aldehyde dehydrogenases reveal structural and functional roles of K⁺-binding sites.
Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K(+)-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K(+)-dependent and K(+)-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K(+) ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K(+) ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA(+)) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent T(m) values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased β-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of β-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, México, Distrito Federal, México.
Georgina
G
Garza-Ramos
Carlos
C
Mújica-Jiménez
Rosario A
RA
Muñoz-Clares
D001426:Bacterial Proteins / Q000737:chemistry*; D052160:Benzothiazoles; D050728:Betaine-Aldehyde Dehydrogenase / Q000737:chemistry*; D001665:Binding Sites; D002414:Cations, Monovalent; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000737:chemistry*; D011188:Potassium / Q000737:chemistry*; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins; D018724:Spinacia oleracea / Q000737:chemistry* / Q000201:enzymology; D019789:Tetraethylammonium / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry
D001426:Bacterial Proteins; D052160:Benzothiazoles; D002414:Cations, Monovalent; D010940:Plant Proteins; D011994:Recombinant Proteins; D013844:Thiazoles; C009462:thioflavin T; D019789:Tetraethylammonium; D050728:Betaine-Aldehyde Dehydrogenase; D011188:Potassium