Stability, unfolding, and structural changes of cofactor-free 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase.
Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.
Beermann
Bernd
B
Institut für Physikalische Chemie der WWU Münster, Corrensstrasse 30 and CeNTech, Heisenbergstrasse 11, 48149 Münster, Germany.
Guddorf
Jessica
J
Boehm
Kristian
K
Albers
Alexander
A
Kolkenbrock
Stephan
S
Fetzner
Susanne
S
Hinz
H-J
HJ
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003720:Densitometry; D049308:Dioxygenases / Q000737:chemistry*; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008027:Light; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001051:Apoenzymes; D002021:Buffers; D006639:Histidine; C101932:1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming); D049308:Dioxygenases