Double Mutant Chimera Q165E/S254K-X21_(TCT) (3ftn)

The NADP⁺-dependent alcohol dehydrogenases (EC 1.1.1.2) from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) are [1] (monomers are colored in different colors) secondary alcohol dehydrogenases. Each of these alcohol dehydrogenases consists of two domains: the (residues 154−294 for TbADH) and the (residues 1−153 and 295−351 for TbADH; contains Zn²⁺ at the active site) separated by a deep cleft. Although, all these three ADHs revealed a high degree of sequence conservation (62-75% identity), them significantly differ in thermostability. The cofactor-binding domains (residues 153−295) of TbADH, CbADH, and EhADH1 were mutually and 3 corresponding chimeras were constructed. The cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH (chimera Χ21_(TCT), 3fsr). This domain replacement significantly destabilized the parent thermophilic enzyme (ΔT_1/2 = −18 °C). But the reverse exchange in CbADH (chimera Χ22_(CTC), 3fpl), had little effect on the thermal stability of the parent mesophilic protein. The exchange of the cofactor-binding domain of TbADH with the homologous domain of EhADH1 (chimera Χ23_(TET), 3fpc) substantially reduced the thermal stability of the thermophilic ADH (ΔT_1/2 = −51 °C) and interfered the oligomerization of the enzyme.

The double mutant of the chimera Χ21_(TCT) (cofactor-binding domain of thermophilic TbADH replaced by that of mesophilic CbADH) Q165E/S254K-X21_(TCT) (3ftn) was constructed by site-directed mutagenesis. In both TbADH and CbADH, Lys257 and Asp237 form an intrasubunit ion pair, in TbADH, Asp237 is also involved in an ion pair bridge with Arg304 of the adjacent monomer. In addition, Arg304 forms intersubunit salt bridge with Glu165 of the first monomer. Therefore, a involving Lys257, Asp237, and Glu165 of one monomer and Arg304 of the adjacent one is present in TbADH (the names of monomers are in brackets). However in mesophilic CbADH (and, therefore, in the chimera Χ21_(TCT), 3fsr) the Gln is situated in position 165 (instead Glu of TbADH) and Met in position 304 (instead Arg of TbADH), so, such an ion pair network does not exist. In the double mutant Q165E/S254K-X21_(TCT) reverse mutation Q165E reconstructs this network (as in parent thermophilic TbADH) that led to significant enhancement of the thermal stability of CbADH (ΔT_1/2^{60 min} = 5.4 °C). Chimera X21_(TCT) (3fsr) is colored magenta and the double mutant Q165E/S254K-X21_(TCT) cyan (3ftn). In chimera X21_(TCT), position 254 is occupied by Ser (due to sequence of exchanged domain). The replacement of Ser254 of CbADH with Lys significantly enhances the stability of the enzyme, due to the formation of . However, this replacing of Ser254 by Lys had a negligible effect on the thermal stability, in contrast to mutation Q165E mentioned above.