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. 2000 May;9(5):991–1001. doi: 10.1110/ps.9.5.991

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

Q N Cao 1, M Stubbs 1, K Q Ngo 1, M Ward 1, A Cunningham 1, E F Pai 1, G C Tu 1, T Hofmann 1
PMCID: PMC2144643  PMID: 10850809

Abstract

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

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Selected References

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  1. Abad-Zapatero C., Rydel T. J., Neidhart D. J., Luly J., Erickson J. W. Inhibitor binding induces structural changes in porcine pepsin. Adv Exp Med Biol. 1991;306:9–21. doi: 10.1007/978-1-4684-6012-4_2. [DOI] [PubMed] [Google Scholar]
  2. Allen B., Blum M., Cunningham A., Tu G. C., Hofmann T. A ligand-induced, temperature-dependent conformational Change in penicillopepsin. Evidence from nonlinear Arrhenius plots and from circular dichroism studies. J Biol Chem. 1990 Mar 25;265(9):5060–5065. [PubMed] [Google Scholar]
  3. Arruda L. K., Vailes L. D., Mann B. J., Shannon J., Fox J. W., Vedvick T. S., Hayden M. L., Chapman M. D. Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases. J Biol Chem. 1995 Aug 18;270(33):19563–19568. doi: 10.1074/jbc.270.33.19563. [DOI] [PubMed] [Google Scholar]
  4. Asakura T., Watanabe H., Abe K., Arai S. Rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases. Eur J Biochem. 1995 Aug 15;232(1):77–83. doi: 10.1111/j.1432-1033.1995.tb20783.x. [DOI] [PubMed] [Google Scholar]
  5. Bailey D., Cooper J. B., Veerapandian B., Blundell T. L., Atrash B., Jones D. M., Szelke M. X-ray-crystallographic studies of complexes of pepstatin A and a statine-containing human renin inhibitor with endothiapepsin. Biochem J. 1993 Jan 15;289(Pt 2):363–371. doi: 10.1042/bj2890363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Balbaa M., Cunningham A., Hofmann T. Secondary substrate binding in aspartic proteinases: contributions of subsites S3 and S'2 to kcat. Arch Biochem Biophys. 1993 Nov 1;306(2):297–303. doi: 10.1006/abbi.1993.1515. [DOI] [PubMed] [Google Scholar]
  7. Baldwin E. T., Bhat T. N., Gulnik S., Hosur M. V., Sowder R. C., 2nd, Cachau R. E., Collins J., Silva A. M., Erickson J. W. Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6796–6800. doi: 10.1073/pnas.90.14.6796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ballance D. J., Buxton F. P., Turner G. Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun. 1983 Apr 15;112(1):284–289. doi: 10.1016/0006-291x(83)91828-4. [DOI] [PubMed] [Google Scholar]
  9. Becker M. M., Harrop S. A., Dalton J. P., Kalinna B. H., McManus D. P., Brindley P. J. Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation. J Biol Chem. 1995 Oct 13;270(41):24496–24501. doi: 10.1074/jbc.270.41.24496. [DOI] [PubMed] [Google Scholar]
  10. Berka R. M., Barnett C. C. The development of gene expression systems for filamentous fungi. Biotechnol Adv. 1989;7(2):127–154. doi: 10.1016/0734-9750(89)90356-x. [DOI] [PubMed] [Google Scholar]
  11. Berka R. M., Ward M., Wilson L. J., Hayenga K. J., Kodama K. H., Carlomagno L. P., Thompson S. A. Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene. 1990 Feb 14;86(2):153–162. doi: 10.1016/0378-1119(90)90274-u. [DOI] [PubMed] [Google Scholar]
  12. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  13. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  14. Blum M., Cunningham A., Bendiner M., Hofmann T. Penicillopepsin, the aspartic proteinase from Penicillium janthinellum: substrate-binding effects and intermediates in transpeptidation reactions. Biochem Soc Trans. 1985 Dec;13(6):1044–1046. doi: 10.1042/bst0131044. [DOI] [PubMed] [Google Scholar]
  15. Campbell E. I., Unkles S. E., Macro J. A., van den Hondel C., Contreras R., Kinghorn J. R. Improved transformation efficiency of Aspergillus niger using the homologous niaD gene for nitrate reductase. Curr Genet. 1989 Jul;16(1):53–56. doi: 10.1007/BF00411084. [DOI] [PubMed] [Google Scholar]
  16. Chen L., Erickson J. W., Rydel T. J., Park C. H., Neidhart D., Luly J., Abad-Zapatero C. Structure of a pepsin/renin inhibitor complex reveals a novel crystal packing induced by minor chemical alterations in the inhibitor. Acta Crystallogr B. 1992 Aug 1;48(Pt 4):476–488. doi: 10.1107/s0108768192001939. [DOI] [PubMed] [Google Scholar]
  17. Choi G. H., Pawlyk D. M., Rae B., Shapira R., Nuss D. L. Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica. Gene. 1993 Mar 30;125(2):135–141. doi: 10.1016/0378-1119(93)90320-3. [DOI] [PubMed] [Google Scholar]
  18. Cunningham A., Hofmann M. I., Hofmann T. Rate-determining steps in penicillopepsin-catalysed reactions. FEBS Lett. 1990 Dec 10;276(1-2):119–122. doi: 10.1016/0014-5793(90)80522-k. [DOI] [PubMed] [Google Scholar]
  19. Cutfield S. M., Dodson E. J., Anderson B. F., Moody P. C., Marshall C. J., Sullivan P. A., Cutfield J. F. The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors. Structure. 1995 Nov 15;3(11):1261–1271. doi: 10.1016/s0969-2126(01)00261-1. [DOI] [PubMed] [Google Scholar]
  20. D'Hondt K., Stack S., Gutteridge S., Vandekerckhove J., Krebbers E., Gal S. Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus. Plant Mol Biol. 1997 Jan;33(1):187–192. doi: 10.1023/a:1005794917200. [DOI] [PubMed] [Google Scholar]
  21. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Foundling S. I., Cooper J., Watson F. E., Cleasby A., Pearl L. H., Sibanda B. L., Hemmings A., Wood S. P., Blundell T. L., Valler M. J. High resolution X-ray analyses of renin inhibitor-aspartic proteinase complexes. 1987 May 28-Jun 3Nature. 327(6120):349–352. doi: 10.1038/327349a0. [DOI] [PubMed] [Google Scholar]
  23. Fruton J. S. The mechanism of the catalytic action of pepsin and related acid proteinases. Adv Enzymol Relat Areas Mol Biol. 1976;44:1–36. doi: 10.1002/9780470122891.ch1. [DOI] [PubMed] [Google Scholar]
  24. Fujinaga M., Chernaia M. M., Tarasova N. I., Mosimann S. C., James M. N. Crystal structure of human pepsin and its complex with pepstatin. Protein Sci. 1995 May;4(5):960–972. doi: 10.1002/pro.5560040516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Garber R. C., Yoder O. C. Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal Biochem. 1983 Dec;135(2):416–422. doi: 10.1016/0003-2697(83)90704-2. [DOI] [PubMed] [Google Scholar]
  26. Groves M. R., Dhanaraj V., Badasso M., Nugent P., Pitts J. E., Hoover D. J., Blundell T. L. A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure. Protein Eng. 1998 Oct;11(10):833–840. doi: 10.1093/protein/11.10.833. [DOI] [PubMed] [Google Scholar]
  27. Harrop S. A., Prociv P., Brindley P. J. Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum. Biochem Biophys Res Commun. 1996 Oct 3;227(1):294–302. doi: 10.1006/bbrc.1996.1503. [DOI] [PubMed] [Google Scholar]
  28. Hofmann T., Allen B., Bendiner M., Blum M., Cunningham A. Effect of secondary substrate binding in penicillopepsin: contributions of subsites S3 and S2' to kcat. Biochemistry. 1988 Feb 23;27(4):1140–1146. doi: 10.1021/bi00404a010. [DOI] [PubMed] [Google Scholar]
  29. Hofmann T. Penicillopepsin. Methods Enzymol. 1976;45:434–452. doi: 10.1016/s0076-6879(76)45038-3. [DOI] [PubMed] [Google Scholar]
  30. Horiuchi H., Yanai K., Okazaki T., Takagi M., Yano K. Isolation and sequencing of a genomic clone encoding aspartic proteinase of Rhizopus niveus. J Bacteriol. 1988 Jan;170(1):272–278. doi: 10.1128/jb.170.1.272-278.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hsu I. N., Delbaere L. T., James M. N., Hofmann T. Penicillopepsin from Penicillium janthinellum crystal structure at 2.8 A and sequence homology with porcine pepsin. Nature. 1977 Mar 10;266(5598):140–145. doi: 10.1038/266140a0. [DOI] [PubMed] [Google Scholar]
  32. James M. N., Sielecki A. R., Hayakawa K., Gelb M. H. Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides. Biochemistry. 1992 Apr 21;31(15):3872–3886. doi: 10.1021/bi00130a019. [DOI] [PubMed] [Google Scholar]
  33. James M. N., Sielecki A. R. Structure and refinement of penicillopepsin at 1.8 A resolution. J Mol Biol. 1983 Jan 15;163(2):299–361. doi: 10.1016/0022-2836(83)90008-6. [DOI] [PubMed] [Google Scholar]
  34. James M. N., Sielecki A., Salituro F., Rich D. H., Hofmann T. Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6137–6141. doi: 10.1073/pnas.79.20.6137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jarai G., van den Hombergh H., Buxton F. P. Cloning and characterization of the pepE gene of Aspergillus niger encoding a new aspartic protease and regulation of pepE and pepC. Gene. 1994 Aug 5;145(2):171–178. doi: 10.1016/0378-1119(94)90002-7. [DOI] [PubMed] [Google Scholar]
  36. Koelsch G., Mares M., Metcalf P., Fusek M. Multiple functions of pro-parts of aspartic proteinase zymogens. FEBS Lett. 1994 Apr 18;343(1):6–10. doi: 10.1016/0014-5793(94)80596-2. [DOI] [PubMed] [Google Scholar]
  37. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997 Jan;10(1):1–6. doi: 10.1093/protein/10.1.1. [DOI] [PubMed] [Google Scholar]
  38. Panthier J. J., Dreyfus M., Roux T. L., Rougeon F. Mouse kidney and submaxillary gland renin genes differ in their 5' putative regulatory sequences. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5489–5493. doi: 10.1073/pnas.81.17.5489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parris K. D., Hoover D. J., Damon D. B., Davies D. R. Synthesis and crystallographic analysis of two rhizopuspepsin inhibitor complexes. Biochemistry. 1992 Sep 8;31(35):8125–8141. doi: 10.1021/bi00150a004. [DOI] [PubMed] [Google Scholar]
  40. Razanamparany V., Jara P., Legoux R., Delmas P., Msayeh F., Kaghad M., Loison G. Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica. Curr Genet. 1992 May;21(6):455–461. doi: 10.1007/BF00351655. [DOI] [PubMed] [Google Scholar]
  41. Sali A., Veerapandian B., Cooper J. B., Foundling S. I., Hoover D. J., Blundell T. L. High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme. EMBO J. 1989 Aug;8(8):2179–2188. doi: 10.1002/j.1460-2075.1989.tb08340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sali A., Veerapandian B., Cooper J. B., Moss D. S., Hofmann T., Blundell T. L. Domain flexibility in aspartic proteinases. Proteins. 1992 Feb;12(2):158–170. doi: 10.1002/prot.340120209. [DOI] [PubMed] [Google Scholar]
  43. Sampath-Kumar P. S., Fruton J. S. Studies on the extended active sites of acid proteinases. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1070–1072. doi: 10.1073/pnas.71.4.1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  46. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  47. Suguna K., Padlan E. A., Bott R., Boger J., Parris K. D., Davies D. R. Structures of complexes of rhizopuspepsin with pepstatin and other statine-containing inhibitors. Proteins. 1992 Jul;13(3):195–205. doi: 10.1002/prot.340130303. [DOI] [PubMed] [Google Scholar]
  48. Suguna K., Padlan E. A., Smith C. W., Carlson W. D., Davies D. R. Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7009–7013. doi: 10.1073/pnas.84.20.7009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tang J., James M. N., Hsu I. N., Jenkins J. A., Blundell T. L. Structural evidence for gene duplication in the evolution of the acid proteases. Nature. 1978 Feb 16;271(5646):618–621. doi: 10.1038/271618a0. [DOI] [PubMed] [Google Scholar]
  50. Tonouchi N., Shoun H., Uozumi T., Beppu T. Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus. Nucleic Acids Res. 1986 Oct 10;14(19):7557–7568. doi: 10.1093/nar/14.19.7557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ward M., Wilson L. J., Kodama K. H. Use of Aspergillus overproducing mutants, cured for integrated plasmid, to overproduce heterologous proteins. Appl Microbiol Biotechnol. 1993 Aug;39(6):738–743. doi: 10.1007/BF00164459. [DOI] [PubMed] [Google Scholar]

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