The goals of this research program have been
- to identify, characterize, clone, sequence, and overexpress adh genes from a variety of hyperthermophilic, thermophilic, mesophilic, and psychrophilic microorganisms;
- to determine the three dimensional structures of these ADHs by X-ray crystallography;
- to analyze and compare the detailed three dimensional structures of the enzymes and to predict structural features conferring thermal stability in the thermophilic enzyme;
- to test the validity of the predictions by altering the mesophilic enzymes to include the relevant features of the thermophilic enzyme using site-directed mutagenesis.
To this end, we compared the molecular properties of five highly homologous ADHs: one from the extreme thermophile Thermoanaerobacter brockii (TbADH) and the others from the pseudo-thermophiles and mesophiles Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH) and Entamoeba invadens (EiADH). All four highly homologous ADHs (60%-76% sequence identity and 72%-86% sequence similarity) are tetrameric, medium-chain, NADP-linked, zinc-containing enzymes with similarly broad substrate ranges. A fifth highly homologous ADH (62% identity) from Mycoplasma pneumoniae (MpADH), was also partially characterized.
The structural genes encoding TbADH and it mesophilic counterparts, CbADH, EhADH, EiADH and MpADH were cloned, sequenced, and overexpressed in Escherichia coli, purified, crystallized, and their 3D structures were determined (in collaboration with the late Prof. Felix Frolow). The 3-D structures of these proteins were the first to be determined for a prokaryotic ADH, as well as for an NADP(H)-dependent ADH
Characterization of the active-site metal ion and the associated ligand amino acids, using atomic absorption spectroscopy analysis and site-directed mutagenesis, verified the presence of a catalytic zinc and the absence of a structural metal ion in TbADH, as well as the involvement of Cys-37, His-59, and Asp-150 (and their analogous amino acid in CbADA, EhADH and MpADH) in Zinc coordination
Site directed mutagenesis studies have been conducted to probe the role of prolines, salt bridges, hydrophobic interactions, ion-pair networks and beta-turns in the thermal stability of these ADHs.