Principles of protein thermostability have been
studied by comparing structures of thermostable proteins
with mesophilic counterparts that have a high degree of
sequence identity. Two tetrameric NADP(H)-dependent alcohol
dehydrogenases, one from Clostridium beijerinckii
(CBADH) and the other from Thermoanaerobacter brockii
(TBADH), having exceptionally high (75%) sequence identity,
differ by 30° in their melting temperatures. The crystal
structures of CBADH and TBADH in their holo-enzyme form
have been determined at a resolution of 2.05 and 2.5 Å,
respectively. Comparison of these two very similar structures
(RMS difference in Cα = 0.8 Å) revealed several
features that can account for the higher thermal stability
of TBADH. These include additional ion pairs, “charged-neutral”
hydrogen bonds, and prolines as well as improved stability
of α-helices and tighter molecular packing. However,
a deeper structural insight, based on the location of stabilizing
elements, suggests that enhanced thermal stability of TBADH
is due mainly to the strategic placement of structural
determinants at positions that strengthen the interface
between its subunits. This is also supported by mutational
analysis of structural elements at critical locations.
Thus, it is the reinforcement of the quaternary structure
that is most likely to be a primary factor in preserving
enzymatic activity of this oligomeric bacterial ADH at
elevated temperatures.