Stability, Unfolding, and Structural Changes of Cofactor-Free 1H-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

• Published in: Biochemistry (Easton) Vol. 46; no. 14; pp. 4241 - 4249
• Main Authors: Beermann, Bernd, Guddorf, Jessica, Boehm, Kristian, Albers, Alexander, Kolkenbrock, Stephan, Fetzner, Susanne, Hinz, H.-J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.04.2007
• Subjects: Amino Acid Sequence; Apoenzymes - chemistry; Buffers; Calorimetry, Differential Scanning; Circular Dichroism; Densitometry; Dioxygenases - chemistry; Enzyme Stability; Histidine - chemistry; Histidine - isolation & purification; Hot Temperature; Hydrogen-Ion Concentration; Light; Models, Chemical; Molecular Sequence Data; Molecular Weight; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Scattering, Radiation; Sequence Homology, Amino Acid; Spectrophotometry, Ultraviolet; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0622423

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 °C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of α-helical structure of ∼60%, based on deconvolution of CD spectra. The percentage of β-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the α/β-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, αP, of the three states and exhibits a significantly lower percentage of α-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 β-type motifs.