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A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase.

Goihberg E, Dym O, Tel-Or S, Levin I, Peretz M, Burstein Y
Proteins 66 196-204 (2007)
Cited: 28 times
EuropePMC logo PMID: 17063493

Abstract

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.

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  1. Function prediction from networks of local evolutionary similarity in protein structure. Erdin S, Venner E, Lisewski AM, Lichtarge O. BMC Bioinformatics 14 Suppl 3 S6 (2013)


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  1. Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA. Appl Environ Microbiol 78 3458-3464 (2012)
  2. Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation. Tian J, Wang P, Gao S, Chu X, Wu N, Fan Y. FEBS J 277 4901-4908 (2010)
  3. A novel scoring function for discriminating hyperthermophilic and mesophilic proteins with application to predicting relative thermostability of protein mutants. Li Y, Middaugh CR, Fang J. BMC Bioinformatics 11 62 (2010)
  4. Thermostability improvement of a streptomyces xylanase by introducing proline and glutamic acid residues. Wang K, Luo H, Tian J, Turunen O, Huang H, Shi P, Hua H, Wang C, Wang S, Yao B. Appl Environ Microbiol 80 2158-2165 (2014)
  5. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Hawwa R, Aikens J, Turner RJ, Santarsiero BD, Mesecar AD. Arch Biochem Biophys 488 109-120 (2009)
  6. A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea. Sayed A, Ghazy MA, Ferreira AJ, Setubal JC, Chambergo FS, Ouf A, Adel M, Dawe AS, Archer JA, Bajic VB, Siam R, El-Dorry H. J Biol Chem 289 1675-1687 (2014)
  7. Characterization of a zinc-containing alcohol dehydrogenase with stereoselectivity from the hyperthermophilic archaeon Thermococcus guaymasensis. Ying X, Ma K. J Bacteriol 193 3009-3019 (2011)
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  11. Design of an activity and stability improved carbonyl reductase from Candida parapsilosis. Jakoblinnert A, van den Wittenboer A, Shivange AV, Bocola M, Heffele L, Ansorge-Schumacher M, Schwaneberg U. J Biotechnol 165 52-62 (2013)
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  13. Low Temperature Adaptation Is Not the Opposite Process of High Temperature Adaptation in Terms of Changes in Amino Acid Composition. Yang LL, Tang SK, Huang Y, Zhi XY. Genome Biol Evol 7 3426-3433 (2015)
  14. Improved thermostability of an acidic xylanase from Aspergillus sulphureus by combined disulphide bridge introduction and proline residue substitution. Yang W, Yang Y, Zhang L, Xu H, Guo X, Yang X, Dong B, Cao Y. Sci Rep 7 1587 (2017)
  15. Seven N-terminal residues of a thermophilic xylanase are sufficient to confer hyperthermostability on its mesophilic counterpart. Zhang S, He Y, Yu H, Dong Z. PLoS One 9 e87632 (2014)
  16. Engineering and kinetic stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase. Jaliani HZ, Farajnia S, Mohammadi SA, Barzegar A, Talebi S. Appl Biochem Biotechnol 171 1805-1818 (2013)
  17. Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution. Goihberg E, Dym O, Tel-Or S, Shimon L, Frolow F, Peretz M, Burstein Y. Proteins 72 711-719 (2008)
  18. Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation. Liao ML, Somero GN, Dong YW. Proc Natl Acad Sci U S A 116 679-688 (2019)
  19. Stability of cytochromes c' from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure. Suka A, Oki H, Kato Y, Kawahara K, Ohkubo T, Maruno T, Kobayashi Y, Fujii S, Wakai S, Lisdiana L, Sambongi Y. Extremophiles 23 239-248 (2019)
  20. Site-directed mutagenesis of coenzyme-independent carotenoid oxygenase CSO2 to enhance the enzymatic synthesis of vanillin. Yao X, Lv Y, Yu H, Cao H, Wang L, Wen B, Gu T, Wang F, Sun L, Xin F. Appl Microbiol Biotechnol 104 3897-3907 (2020)
  21. Without salt, the 'thermophilic' protein Mth10b is just mesophilic. Zhang N, Pan XM, Ge M. PLoS One 7 e53125 (2012)
  22. Case Reports Improving the catalytic thermostability of Bacillus altitudinis W3 ω-transaminase by proline substitutions. Xie Z, Zhai L, Meng D, Tian Q, Guan Z, Cai Y, Liao X. 3 Biotech 10 323 (2020)
  23. Regulation of enzyme activity of alcohol dehydrogenase through its interactions with pyruvate-ferredoxin oxidoreductase in Thermoanaerobacter tengcongensis. Wang Q, Wang Q, Tong W, Bai X, Chen Z, Zhao J, Zhang J, Liu S. Biochem Biophys Res Commun 417 1018-1023 (2012)
  24. A Thermally Stable Protein EPP1 of Corn Borer Ostrinia furnacalis Regulates Hemocytic Encapsulation. Hu J, Feng X, Yao L, Meng M, Du Y, Dong Y, Song Z, Tian M, Chen Y. J Innate Immun 13 280-294 (2021)
  25. Chemical modification for improving catalytic performance of lipase B from Candida antarctica with hydrophobic proline ionic liquid. Zhang XG, Xue Y, Lu ZP, Xu HJ, Hu Y. Bioprocess Biosyst Eng 45 749-759 (2022)
  26. Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus. Yoshimi T, Fujii S, Oki H, Igawa T, Adams HR, Ueda K, Kawahara K, Ohkubo T, Hough MA, Sambongi Y. Acta Crystallogr F Struct Biol Commun 78 217-225 (2022)
  27. Enhancing the thermostability of Streptomyces cyaneofuscatus strain Ms1 tyrosinase by multi-factors rational design and molecular dynamics simulations. Li Z, Zhao C, Li D, Wang L. PLoS One 18 e0288929 (2023)


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