Key Structural Elements of Thermal Stability in the Thermophilic Alcohol Dehydrogenase from Thermoanaerobacter brockii
Oren BOGIN*a' Moshe PERETZa, Yakov KORKHINb, Yael HACHAMa, A. J. KALB(Gilboa)b, Felix FROLOWc, and Yigal BURSTEINa
a Departments of Organic Chemistry, The Weizman Institute of Science, Rehovot 76100, Israel
b Structural Biology, The Weizman Institute of Science, Rehovot 76100, Israel
c Chemical Services, The Weizman Institute of Science, Rehovot 76100, Israel
The molecular basis for thermophily remains an enigma. Elucidation of the mechanisms of thermophily will depend heavily on the understanding of the nature of the differences in thermostability of families of highly homologous proteins.
Our laboratory has been studying a family of structurally and enzymatically closely related alcohol dehydrogenases (ADHs). The genes encoding the thermophilic Thermoanaerobacter brockii ADH (TBADH) and the mesophilic Clostridium beijerinckii ADH (CBADH) have been isolated, characterized, sequenced, cloned, over-expressed in E. coli, and the enzymes were crystallized and their three dimensional structures determined at resolutions of 2.5Å and 2.05Å, (PDB entries, 1 YKF and 1 PED), respectively. Although the enzymes share 75% amino acid sequence identity, they differ greatly in their thermal stability. TBADH shows T1/260 min (temperature at which 50% of the enzymatic activity is lost after 60 min) and Tm (melting temperature by differential scanning calorimetry) values of 93.3℃ and 98.5℃, respectively, while CBADH has a rl/2ョ min value of 63.3℃ and a Tm of 66℃. Detailed comparison of their high resolution X-ray structures suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants that strengthen the interface between its subunits, and preserve its oligomeric form at high temperatures. Using sitedirected mutagenesis, the structural elements involved in inter-subunit interactions in TBADH, have been incorporated into CBADH. Introducing the inter-subunit iron-pair Q 165E/M304R, increases the Tm of CBADH by 14.4℃ and a double mutation in the region of an inter-subunit hydrophobic patch (A273V/L275P) increases the T1/260min by 7℃. Replacing Trp90, located at the hydrophobic inter-dimer interface of TBADH with Asp, dissociated the tetrameric enzyme into mesopilic, enzymatically inactive dimers. Less pronounced effects on the thermo-stabilization of CBADH were observed when structural elements of intra-subunit interactions, and some proline residues positioned at critical sites in TBADH, have been incorporated into CBADH.
We, therefore, suggest that it is the reinforecement of the quaternary structure that is most likely to preserve the enzymatic activity of the oligometic TBADH at elevated temperatures.
