Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Abstract

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K-and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R-and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.Keywords: Alcohol dehydrogenase; Clostridium beijerinckii; Thermoanaerobacter brockii; site-directed mutagenesis; ion-pair network; salt bridge; thermostability Discerning and manipulating the structurally different elements of mesophilic and thermophilic members of homologous protein families is the key to understanding the adaptation of hyperthermophilic proteins to extreme temperatures (for review, see Kumar et al. 2000bKumar et al. , 2001Szilagyi and Zavodszky 2000;Sterner and Liebl 2001 These authors contributed equally to the present work.

Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis

“…On the other hand, proline composition increased significantly, which might be the structural base of the rigidity of hyperthermophilic enzymes (23). The difference between hyperthermophilic and mesophilic proteins/enzymes would provide clues for increasing the thermal stability of mesophilic enzymes (8,9). The physiological role of T. guaymasensis ADH seems not to be clearly ascertained, simply relying on its conserved domain homology to threonine dehydrogenase.…”

A conformationally isoformic thermophilic protein with high kinetic unfolding barriers

“…It has been shown in the thermophilic glutamate dehydrogenase that ion pair networks provide more stability to the protein molecules than isolated ion pairs, and this phenomenon is particularly effective at higher temperatures [34]. The role of salt bridges in the thermal stability of TBADH was demonstrated earlier by Bogin et al [21]. In addition, disruption of an ionic network around Arg20 accelerates the unfolding rate in D-glyceraldehyde-3-phosphate dehydrogenase [35].…”

“…We performed an additional search for salt bridges, using a structure with a slightly higher resolution than in the earlier work (PDB id 1YKF) with the Web version of the WHAT IF software, which revealed 14 intermolecular salt bridges. Indeed, a role of ionic networks for thermal stability has been demonstrated [21]. A closer examination of the structural determinants showed that they are strategically placed at the subunit interface (e.g., Fig.…”

“…This is a surprisingly low value for such a large protein and stands in marked contrast to the known thermostability of TBADH. From the structural analysis, it was evident that ionic interactions play an important role in the stability of TBADH [17,21]. Therefore, to understand the true effect of chemical denaturation, urea was employed because it is a noncharged chaotrope (Fig.…”

A Water Mediated Electrostatic Interaction Gives Thermal Stability to the “Tail” Region of the GrpE Protein from E. coli

“…Ikura and coworkers have shown that there are important water-mediated interactions at the barnase-barstar protein-protein interface (Ikura et al, 2004). Additional research into the significance of water mediated interactions have shown that engineered ion pairs from a protein found in a thermophile when introduced into an homologous protein from a mesophilic bacterium lead to additional water mediated interactions and enhanced stability (Bogin et al, 2002). Another study showed that a new water mediated interaction was present for a protein at lower temperature suggesting the plasticity of ion pairs in proteins (Natesh et al, 2003).…”