Proteolytic Cleavage of Hansenula anomala Flavocytochrome b2 into Its Two Functional Domains

Abstract

The tryptic cleavage of yeast flavocytochrome b2 into its 2 functional domains, a cytochrome b2 core and a flavodehydrogenase, was described previously. The lactate dehydrogenase efficiency of the latter was dramatically low, only .apprx. 1% that of intact flavocytochrome b2. A new flavodehydrogenase derivative of H. anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I was studied. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, .apprx. 80% that of flavocytochrome b2. Evidently, the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. The results are discussed in terms of gene fusion.