Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

Journal of Biochemistry

Volumn 145, Issue 1, 2009, Pages 103-113

  Kusano, Masayuki ^a   Yasukawa, Kiyoshi ^a   Inouye, Kuniyo ^a  

^a KYOTO UNIVERSITY   (Japan)

Author keywords

Active site; Metalloproteinase; Site directed mutagenesis; Stability; Thermolysin

Indexed keywords

ALANINE; AMINO ACID; AMINO ACID DERIVATIVE; ASPARAGINE; ASPARTIC ACID; CASEIN; ENZYME VARIANT; GLYCINE; ISOLEUCINE; N [3 (2 FURYL)ACRYLOYL]GLYCYLLEUCYLAMIDE; N CARBOBENZOXYASPARTYLPHENYLALANYLMETHYL ESTER; PHENYLALANINE; POLYPEPTIDE; SERINE; THERMOLYSIN; TRYPTOPHAN; TYROSINE; UNCLASSIFIED DRUG; VALINE; ZINC ION;

ALPHA HELIX; AMINO ACID SUBSTITUTION; AMINO TERMINAL SEQUENCE; ARTICLE; CARBOXY TERMINAL SEQUENCE; CATALYSIS; ENZYME ACTIVATION; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME INACTIVATION; ENZYME STABILITY; ENZYME SUBSTRATE; ESCHERICHIA COLI; HYDROLYSIS; NONHUMAN; SITE DIRECTED MUTAGENESIS; THERMOSTABILITY; WILD TYPE;

CATALYSIS; CATALYTIC DOMAIN; HYDROGEN-ION CONCENTRATION; HYDROLYSIS; KINETICS; MUTAGENESIS, SITE-DIRECTED; PEPTIDES; THERMOLYSIN;

ESCHERICHIA COLI;

EID: 58149105482     PISSN: 0021924X     EISSN: 17562651     Source Type: Journal    
DOI: 10.1093/jb/mvn140     Document Type: Article

References (51)

1. Endo, S. (1962) Studies on protease produced by thermophilic bacteria. J. Ferment. Technol. 40, 346-353
2. van den Burg, B. and Eijsink, V.G. (2004) Thermolysin in Handbook of Proteolytic Enzymes, 2nd ed. (Barrett, J.A., Rawlings, N.D., and Woessner, J.F., eds.) Vol. 1, pp. 374-387, Elsevier, Amsterdam
3. Inouye, K. (2003) Thermolysin in Handbook of Food Enzymology (Whitaker, J.R., Voragen, A.G.J., and Wong, D.W.S., eds.) pp. 1019-1028, Marcel Dekker, New York
4. Latt, S.A., Holmquist, B., and Vallee, B.L. (1969) Thermolysin: a zinc metalloenzyme. Biochem. Biophys. Res. Commun. 37, 333-339
5. Feder, J., Garrett, L.R., and Wildi, B.S. (1971) Studies on the role of calcium in thermolysin. Biochemistry 10, 4552-4556
6. Tajima, M., Urabe, I., Yutani, K., and Okada, H. (1976) Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease. Eur. J. Biochem. 64, 243-247
7. Morihara, K. and Tsuzuki, H. (1970) Thermolysin: kinetic study with oligopeptides. Eur. J. Biochem. 15, 374-380
8. Inouye, K., Lee, S.-B., and Tonomura, B. (1996) Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts. Biochem. J. 315, 133-138
9. Titani, K., Hermodson, M.A., Ericsson, L.H., Walsh, K.A., and Neurath, H. (1972) Amino-acid sequence of thermolysin. Nature 238, 35-37
10. Holmes, M.A. and Matthews, B.W. (1982) Structure of thermolysin refined at 1.6Å resolution. J. Mol. Biol. 160, 623-639
11. Hangauer, D.G., Monzingo, A.F., and Matthews, B.W. (1984) An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides. Biochemistry 23, 5730-5741
12. Mock, W.L. and Aksamawati, M. (1994) Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis. Biochem. J. 302, 57-68
13. Oyama, K., Kihara, K., and Nonaka, Y. (1981) On the mechanism of the action of thermolysin: kinetic study of the thermolysin-catalysed condensation reaction of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester. J. Chem. Soc. Perkin II, 356-360
14. Inouye, K. (1992) Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin. J. Biochem. 112, 335-340
15. Inouye, K., Kusano, M., Hashida, Y., Minoda, M., and Yasukawa, K. (2007) Engineering, expression, purification, and production of recombinant thermolysin. Biotechnol. Annu. Rev. 13, 43-64
16. Hanzawa, S. and Kidokoro, S. (1999) Thermolysin. in Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation (Flickinger, M.C. and Drew, S.W., eds.) pp. 2527-2535, John Wiley & Sons, New York
17. Inouye, K., Kuzuya, K., and Tonomura, B. (1998) Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity. Biochim. Biophys. Acta. 1388, 209-214
18. Inouye, K., Lee, S.-B., Nambu, K., and Tonomura, B. (1997) Effects of pH, temperature, and alcohols on remarkable activation of thermolysin by salts. J. Biochem. 122, 358-364
19. Oneda, H., Muta, Y., and Inouye, K. (2004) Substrate-dependent activation of thermolysin by salt. Biosci. Biotechnol. Biochem. 68, 1811-1813
20. Inouye, K., Kuzuya, K., and Tonomura, B. (1998) Effect of salts on the solubility of thermolysin: a remarkable increase in the solubility as well as the activity by the addition of salts without aggregation or dispersion of thermolysin. J. Biochem. 123, 847-852
21. Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta. 1774, 1281-1288
22. Matsumiya, Y., Nishikawa, K., Aoshima, H., Inouye, K., and Kubo, M. (2004) Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site. J. Biochem. 135, 547-553
23. Matsumiya, Y., Nishikawa, K., Inouye, K., and Kubo, M. (2005) Mutational effect for stability in a conserved region of thermolysin. Lett. Appl. Microbiol. 40, 329-334
24. Takita, T., Aono, T., Sakurama, H., Itoh, T., Wada, T., Minoda, M., Yasukawa, K., and Inouye, K. (2008) Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability. Biochim. Biophys. Acta. 1784, 481-488
25. Kusano, M., Yasukawa, K., Hashida, Y., and Inouye, K. (2006) Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin. J. Biochem. 139, 1017-1023
26. Tatsumi, C., Hashida, Y., Yasukawa, K., and Inouye, K. (2007) Effects of site-directed mutagenesis of the surface residues Gln128 and Gln225 of thermolysin on its catalytic activity. J. Biochem. 141, 835-842
27. O'Donohue, M.J., Roques, B.P., and Beaumont, A. (1994) Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin. Biochem. J. 300, 599-603
28. Inouye, K., Minoda, M., Takita, T., Sakurama, H., Hashida, Y., Kusano, M., and Yasukawa, K. (2006) Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization. Protein Expr. Purif. 46, 248-255
29. Yasukawa, K., Kusano, M., and Inouye, K. (2007) A new method for the extracellular production of recombinant thermolysin by co-expressing the mature sequence and pro-sequence in Escherichia coli. Protein Eng. Des. Sel. 20, 375-383
30. Feder, J. (1968) A spectrophotometric assay for neutral protease. Biochem. Biophys. Res. Commun. 32, 326-332
31. Yasukawa, K., Kusano, M., Nakamura, K., and Inouye, K. (2006) Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin. Protein Expr. Purif. 46, 332-336
32. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
33. Sakoda, M. and Hiromi, K. (1976) Determination of the best-fit values of kinetic parameters of the Michaelis-Menten equation by the method of least squares with Taylor expansion. J. Biochem. 80, 547-555
34. Eijsink, V.G., van den Burg, B., Vriend, G., Berendsen, H.J., and Venema, G. (1991) Thermostability of Bacillus subtilis neutral protease. Biochem. Int. 24, 517-525
35. Inouye, K., Shimada, T., and Yasukawa, K. (2007) Effects of neutral salts and alcohols on the activity of Streptomyces caespitosus neutral protease. J. Biochem. 142, 317-324
36. Schechter, I and Berger, A. (1967) On the size of the active site in proteases. I. papain. Biochem. Biophys. Res. Commun. 27, 157-162
37. Holland, D.R., Tronrud, D.E., Pley, H.W., Flaherty, K.M., Stark, W., Jansonius, J.N., McKay, D.B., and Matthews, B.W. (1992) Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis. Biochemistry 31, 11310-11316
38. Matthews, B.W. (1988) Structural basis of the action of thermolysin and related zinc peptidases. Acc. Chem. Res. 21, 333-340
39. Pelmenschikov, V., Blomberg, M.R., and Siegbahn, P.E. (2002) A theoretical study of the mechanism for peptide hydrolysis by thermolysin. J. Biol. Inorg. Chem. 7, 284-298
40. Inouye, K., Mazda, N., and Kubo, M. (1998) Need for aromatic residue at position 115 for proteolytic activity found by site-directed mutagenesis of tryptophan 115 in thermolysin. Biosci. Biotechnol. Biochem. 62, 798-800
41. Tronrud, D.E., Monzingo, A.F., and Matthews, B.W. (1986) Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin. Eur. J. Biochem. 157, 261-268
42. Kamo, M., Inouye, K., Nagata, K., and Tanokura, M. (2005) Preliminary X-ray crystallographic analysis of thermolysin in the presence of 4M NaCl. Acta. Cryst. D61, 710-712
43. Durrschmidt, P., Mansfeld, J., and Ulbrich-Hofmann, R. (2005) An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding. FEBS J. 272, 1523-1534
44. Yutani, K., Ogasahara, K., Tsujita, T., and Sugino, Y. (1987) Dependence of conformational stability on hydrophobicity of the amino acid residue in a series of variant proteins substituted at a unique position of tryptophan synthase α subunit. Proc. Natl Acad. Sci. USA 84, 4441-4444
45. Shoichet, B.K., Baase, W.A., Kuroki, R., and Matthews, B.W. (1995) A relationship between protein stability and protein function. Proc. Natl Acad. Sci. USA 92, 452-456
46. Hashida, Y. and Inouye, K. (2007) Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity. J. Biochem. 141, 843-853
47. Hashida, Y. and Inouye, K. (2007) Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin. J. Biochem. 141, 879-888
48. Cherry, J.R. and Fidantsef, A.L. (2003) Directed evolution of industrial enzymes: an update. Curr. Opin. Biotechnol. 14, 438-443
49. Matthews, B.W. (1996) Structural and genetic analysis of the folding and function of T4 lysozyme. FASEB J. 10, 35-41
50. LiCata, V.J. and Ackers, G.K. (1995) Long-range, small magnitude nonadditivity of mutational effects in proteins. Biochemistry 34, 3133-3139
51. de Kreij, A., van den Burg, B., Venema, G., Vriend, G., Eijsink, V.G., and Nielsen, J.E. (2002) The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease. J. Biol. Chem. 277, 15432-15438