Biochemical and Structural Properties of Chimeras Constructed by Exchange of Cofactor-Binding Domains in Alcohol Dehydrogenases from Thermophilic and Mesophilic Microorganisms

摘要

The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from thenthermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckiin(CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Threenchimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADHnwas replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera Χ21(TCT)].nThis domain exchange significantly destabilized the parent thermophilic enzyme (ΔT1/2 =-18 u0002C).nThe reverse exchange in CbADH [chimera Χ22(CTC)], however, had little effect on the thermal stability ofnthe parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with thenhomologous domain of EhADH1 [chimera Χ23(TET)] substantially reduced the thermal stability of thenthermophilic ADH (ΔT1/2 = -51 u0002C) and impeded the oligomerization of the enzyme. All three chimericnproteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structuresnwere determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with thenstructures of their parent ADHs showed no significant changes to their CR chains, suggesting thatnthe difference in the thermal stability of the three parent ADHs and their chimeric mutants could be duento a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces.nIndeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a prolinenresidue at a strategic position in the major horse liver ADH-type dimerization interface (ΔT1/2=35 u0002C) or bynintroduction of intersubunit electrostatic interactions (ΔT1/2 =6 u0002C).