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Protein Sci. 1998 May; 7(5): 1156–1163.
PMCID: PMC2144005

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.

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Selected References

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  • Bogin O, Peretz M, Burstein Y. Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids. Protein Sci. 1997 Feb;6(2):450–458. [PMC free article] [PubMed]
  • Davies GJ, Gamblin SJ, Littlechild JA, Watson HC. The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent. Proteins. 1993 Mar;15(3):283–289. [PubMed]
  • Delboni LF, Mande SC, Rentier-Delrue F, Mainfroid V, Turley S, Vellieux FM, Martial JA, Hol WG. Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions. Protein Sci. 1995 Dec;4(12):2594–2604. [PMC free article] [PubMed]
  • Hardy F, Vriend G, Veltman OR, van der Vinne B, Venema G, Eijsink VG. Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines. FEBS Lett. 1993 Feb 8;317(1-2):89–92. [PubMed]
  • Hennig M, Darimont B, Sterner R, Kirschner K, Jansonius JN. 2.0 A structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability. Structure. 1995 Dec 15;3(12):1295–1306. [PubMed]
  • Hol WG, van Duijnen PT, Berendsen HJ. The alpha-helix dipole and the properties of proteins. Nature. 1978 Jun 8;273(5662):443–446. [PubMed]
  • Horovitz A, Serrano L, Avron B, Bycroft M, Fersht AR. Strength and co-operativity of contributions of surface salt bridges to protein stability. J Mol Biol. 1990 Dec 20;216(4):1031–1044. [PubMed]
  • Ismaiel AA, Zhu CX, Colby GD, Chen JS. Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. J Bacteriol. 1993 Aug;175(16):5097–5105. [PMC free article] [PubMed]
  • Kirino H, Aoki M, Aoshima M, Hayashi Y, Ohba M, Yamagishi A, Wakagi T, Oshima T. Hydrophobic interaction at the subunit interface contributes to the thermostability of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus. Eur J Biochem. 1994 Feb 15;220(1):275–281. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lamed RJ, Zeikus JG. Novel NADP-linked alcohol--aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria. Biochem J. 1981 Apr 1;195(1):183–190. [PMC free article] [PubMed]
  • Matthews BW. Structural and genetic analysis of protein stability. Annu Rev Biochem. 1993;62:139–160. [PubMed]
  • Matthews BW, Nicholson H, Becktel WJ. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6663–6667. [PMC free article] [PubMed]
  • Menéndez-Arias L, Argos P. Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. J Mol Biol. 1989 Mar 20;206(2):397–406. [PubMed]
  • Moriyama H, Onodera K, Sakurai M, Tanaka N, Kirino-Kagawa H, Oshima T, Katsube Y. The crystal structures of mutated 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 and their relationship to the thermostability of the enzyme. J Biochem. 1995 Feb;117(2):408–413. [PubMed]
  • Nicholson H, Tronrud DE, Becktel WJ, Matthews BW. Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme. Biopolymers. 1992 Nov;32(11):1431–1441. [PubMed]
  • Peretz M, Bogin O, Keinan E, Burstein Y. Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii. Int J Pept Protein Res. 1993 Nov;42(5):490–495. [PubMed]
  • Peretz M, Bogin O, Tel-Or S, Cohen A, Li G, Chen JS, Burstein Y. Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii. Anaerobe. 1997 Aug;3(4):259–270. [PubMed]
  • Bogin O, Peretz M, Burstein Y. Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids. Protein Sci. 1997 Feb;6(2):450–458. [PMC free article] [PubMed]
  • Peretz M, Burstein Y. Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii. Biochemistry. 1989 Aug 8;28(16):6549–6555. [PubMed]
  • Perutz MF, Raidt H. Stereochemical basis of heat stability in bacterial ferredoxins and in haemoglobin A2. Nature. 1975 May 15;255(5505):256–259. [PubMed]
  • Querol E, Perez-Pons JA, Mozo-Villarias A. Analysis of protein conformational characteristics related to thermostability. Protein Eng. 1996 Mar;9(3):265–271. [PubMed]
  • Russell RJ, Taylor GL. Engineering thermostability: lessons from thermophilic proteins. Curr Opin Biotechnol. 1995 Aug;6(4):370–374. [PubMed]
  • Tanner JJ, Hecht RM, Krause KL. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry. 1996 Feb 27;35(8):2597–2609. [PubMed]
  • Tomschy A, Böhm G, Jaenicke R. The effect of ion pairs on the thermal stability of D-glyceraldehyde 3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. Protein Eng. 1994 Dec;7(12):1471–1478. [PubMed]
  • Vogt G, Woell S, Argos P. Protein thermal stability, hydrogen bonds, and ion pairs. J Mol Biol. 1997 Jun 20;269(4):631–643. [PubMed]
  • Walker JE, Wonacott AJ, Harris JI. Heat stability of a tetrameric enzyme, D-glyceraldehyde-3-phosphate dehydrogenase. Eur J Biochem. 1980 Jul;108(2):581–586. [PubMed]
  • Warren GL, Petsko GA. Composition analysis of alpha-helices in thermophilic organisms. Protein Eng. 1995 Sep;8(9):905–913. [PubMed]
  • Watanabe K, Chishiro K, Kitamura K, Suzuki Y. Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006. J Biol Chem. 1991 Dec 25;266(36):24287–24294. [PubMed]
  • Watanabe K, Hata Y, Kizaki H, Katsube Y, Suzuki Y. The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization. J Mol Biol. 1997 May 30;269(1):142–153. [PubMed]
  • Watanabe K, Kitamura K, Suzuki Y. Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues. Appl Environ Microbiol. 1996 Jun;62(6):2066–2073. [PMC free article] [PubMed]
  • Watanabe K, Masuda T, Ohashi H, Mihara H, Suzuki Y. Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule. Eur J Biochem. 1994 Dec 1;226(2):277–283. [PubMed]
  • Yip KS, Stillman TJ, Britton KL, Artymiuk PJ, Baker PJ, Sedelnikova SE, Engel PC, Pasquo A, Chiaraluce R, Consalvi V, et al. The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure. 1995 Nov 15;3(11):1147–1158. [PubMed]
  • Zhang Z, Djebli A, Shoham M, Frolow F, Peretz M, Burstein Y. Crystal parameters of an alcohol dehydrogenase from the extreme thermophile Thermoanaerobium brockii. J Mol Biol. 1993 Mar 5;230(1):353–355. [PubMed]

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