Tetrameric alcohol dehydrogenases

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.

The of overall Cα backbone of all these chimeras (rmsd 0.45-0.65 Å) with those of the parent enzymes, did not reveal significant structural changes. So, the differences in the thermal stability of the chimeras and the parent enzymes could be caused by relatively small specific changes located at the important points of the NADP⁺-dependent alcohol dehydrogenases. For example see Cα superposition for the X23_(TET) chimera (red) (3fpc) and its parent ADHs (TbADH, colored blue (1ped), and EhADH1, colored lime (1y9a). The RMSDs of the TbADH−EhADH1, TbADH−Χ23_(TET), and EhADH1−Χ23_(TET) were 0.68, 0.56, and 0.48 Å, respectively.

The 3D structure of CbADH with the substitution Q100P () was solved at 2.25 Å resolution (2b83). The of Gln100 with Pro did not cause significant structural changes in the protein structure. The residues of the wildtype protein are colored lime and the residues of the mutant one in cyan. Only 2 H-bonds were lost, one between Oε1 of Gln100 and the main chain N of Gly297, and the second between Nε2 of Gln100 and the main chain carbonyl O of Gly297. The mutation caused that an additional CH₂ group (Cδ of Pro100) is surrounded by nonpolar residues: Pro88 (3.8 Å), Trp90 (3.5 Å), and Val95 (4 Å). These residues (P100, P88, W90, and V95) are situated on a protruding lobe of the protein. An additional 11 aliphatic and aromatic carbon atoms are situated within the distance of 6 Å from Cδ of Pro100 (two methyl groups of Val95; three carbon atoms of the Trp90 indole group; Cβ and Cγ methylene groups of Pro100; Cβ and Cγ of Gln101, and two carbons of the Phe99 phenyl ring).

Ribbon diagram of the EhADH1 (2oui). Proline residues (ball representation) are colored orange (Pro275) (which is important for thermal stabilization) and cyan (Pro100). of the structures of the wild-type apo-EhADH1 (colored lime, 1y9a) and the apo D275P-EhADH1 mutant (colored orange) (2oui). Pro275 and Asp275 are labeled red. Residues within a distance of 4 Å from the mutation are shown (names of monomers are in brackets). Replacing with significantly enhanced the thermal stability of EhADH1: ΔT_1/2^60min = +9.3°C, ΔT_1/2^CD = +10°C. The reverse mutation in the thermophilic (1ykf; colored magenta) - substitution of wt TbADH Pro275 with (2nvb; colored cyan) reduced the thermal stability of the enzyme: ΔT_1/2^60min = -13.8°C, ΔT_1/2^CD = -18.8°C. Nitrogen and oxygen atoms are colored in CPK colors. Pro275 and Asp275 are labeled red (names of monomers are in brackets). These findings indicate that a single proline mutation is responsible for the significant differences in the thermal stability of ADHs, and show the importance of prolines in the protein stability. It was also shown that substitution by proline at the important positions could significantly stabilize the protein.