Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.
A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.
Bogin
O
O
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Peretz
M
M
Hacham
Y
Y
Korkhin
Y
Y
Frolow
F
F
Kalb(Gilboa)
A J
AJ
Burstein
Y
Y
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D003013:Clostridium / Q000201:enzymology*; D004795:Enzyme Stability; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000201:enzymology*; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D011392:Proline; D000426:Alcohol Dehydrogenase