MOLECULAR-CLONING, NUCLEOTIDE SEQUENCING, AND EXPRESSION OF GENES ENCODING ALCOHOL DEHYDROGENASES FROM THE THERMOPHILE THERMOANAEROBACTER-BROCKII AND THE MESOPHILE CLOSTRIDIUM-BEIJERINCKII

Proteins play a pivotal role in thermophily bacteria. Comparing the mo lecular properties of homologous proteins from thermophilic and mesoph ilic bacteria is important for understanding the mechanisms of microbi al adaptation to extreme environments. The thermophile Thermoanaerobac ter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinc kii contain an NADP(H)-linked, zinc-containing secondary alcohol dehyd rogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, seq uenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 n ucleotides long. The TB adh gene encoded an amino acid sequence identi cal to that of the purified TBADH. Alignment of the deduced;amino acid sequences of the TB and CB adh genes' showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons wit h A or T in the third position. Multiple sequence alignment of ADHs fr om different sources revealed that two (Cys-46 and His-67) of the thre e ligands for the catalytic Zn atom of the horse-liver ADH are preserv ed in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. Th e substrate specificities and thermostabilities of the TBADH and CBADH expressed in E. coil were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the excha nge of hydrophilic and large hydrophobic residues in CBADH for the sma ll hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T brockii enzyme. (C) 1997 Acade mic Press.