Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation.
Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.
Research Centre Applied Biocatalysis and Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Iskandar
I
Dib
Anita
A
Slavica
Waander
W
Riethorst
Bernd
B
Nidetzky
D002511:Cephalosporins / Q000032:analysis / Q000493:pharmacokinetics; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D004718:Saccharomycetales / Q000201:enzymology*; D013816:Thermodynamics
D002511:Cephalosporins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D003605:D-Amino-Acid Oxidase