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. 1999 May 1;339(Pt 3):497–502.

Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity.

K M Fukasawa 1, K Fukasawa 1, M Harada 1, J Hirose 1, T Izumi 1, T Shimizu 1
PMCID: PMC1220182  PMID: 10215585

Abstract

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.

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Selected References

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  1. Baset H. A., Ford-Hutchinson A. W., O'Neill G. P. Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally related to mammalian leukotriene A4 hydrolases. J Biol Chem. 1998 Oct 23;273(43):27978–27987. doi: 10.1074/jbc.273.43.27978. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Gomis-Rüth F. X., Stöckler W. Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the 'metzincins'. FEBS Lett. 1993 Sep 27;331(1-2):134–140. doi: 10.1016/0014-5793(93)80312-i. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cadel S., Foulon T., Viron A., Balogh A., Midol-Monnet S., Noël N., Cohen P. Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2963–2968. doi: 10.1073/pnas.94.7.2963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cadel S., Pierotti A. R., Foulon T., Créminon C., Barré N., Segrétain D., Cohen P. Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules. Mol Cell Endocrinol. 1995 Apr 28;110(1-2):149–160. doi: 10.1016/0303-7207(95)03529-g. [DOI] [PubMed] [Google Scholar]
  6. Fukasawa K. M., Fukasawa K., Kanai M., Fujii S., Harada M. Molecular cloning and expression of rat liver aminopeptidase B. J Biol Chem. 1996 Nov 29;271(48):30731–30735. doi: 10.1074/jbc.271.48.30731. [DOI] [PubMed] [Google Scholar]
  7. HOPSU V. K., KANTONEN U. M., GLENNER G. G. A PEPTIDASE FROM RAT TISSUES SELECTIVELY HYDROLYZING N-TERMINAL ARGININE AND LYSINE RESIDUES. Life Sci. 1964 Dec;3:1449–1453. doi: 10.1016/0024-3205(64)90087-6. [DOI] [PubMed] [Google Scholar]
  8. Haeggström J. Z., Wetterholm A., Vallee B. L., Samuelsson B. Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity. Biochem Biophys Res Commun. 1990 Nov 30;173(1):431–437. doi: 10.1016/s0006-291x(05)81076-9. [DOI] [PubMed] [Google Scholar]
  9. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  10. Hooper N. M. Families of zinc metalloproteases. FEBS Lett. 1994 Oct 31;354(1):1–6. doi: 10.1016/0014-5793(94)01079-x. [DOI] [PubMed] [Google Scholar]
  11. Jongeneel C. V., Bouvier J., Bairoch A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 1989 Jan 2;242(2):211–214. doi: 10.1016/0014-5793(89)80471-5. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Medina J. F., Wetterholm A., Rådmark O., Shapiro R., Haeggström J. Z., Vallee B. L., Samuelsson B. Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7620–7624. doi: 10.1073/pnas.88.17.7620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Minami M., Bito H., Ohishi N., Tsuge H., Miyano M., Mori M., Wada H., Mutoh H., Shimada S., Izumi T. Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297. FEBS Lett. 1992 Sep 14;309(3):353–357. doi: 10.1016/0014-5793(92)80806-r. [DOI] [PubMed] [Google Scholar]
  15. Minami M., Ohishi N., Mutoh H., Izumi T., Bito H., Wada H., Seyama Y., Toh H., Shimizu T. Leukotriene A4 hydrolase is a zinc-containing aminopeptidase. Biochem Biophys Res Commun. 1990 Dec 14;173(2):620–626. doi: 10.1016/s0006-291x(05)80080-4. [DOI] [PubMed] [Google Scholar]
  16. Minami M., Ohno S., Kawasaki H., Rådmark O., Samuelsson B., Jörnvall H., Shimizu T., Seyama Y., Suzuki K. Molecular cloning of a cDNA coding for human leukotriene A4 hydrolase. Complete primary structure of an enzyme involved in eicosanoid synthesis. J Biol Chem. 1987 Oct 15;262(29):13873–13876. [PubMed] [Google Scholar]
  17. Mueller M. J., Blomster M., Oppermann U. C., Jörnvall H., Samuelsson B., Haeggström J. Z. Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5931–5935. doi: 10.1073/pnas.93.12.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohishi N., Izumi T., Minami M., Kitamura S., Seyama Y., Ohkawa S., Terao S., Yotsumoto H., Takaku F., Shimizu T. Leukotriene A4 hydrolase in the human lung. Inactivation of the enzyme with leukotriene A4 isomers. J Biol Chem. 1987 Jul 25;262(21):10200–10205. [PubMed] [Google Scholar]
  19. Olsen J., Cowell G. M., Kønigshøfer E., Danielsen E. M., Møller J., Laustsen L., Hansen O. C., Welinder K. G., Engberg J., Hunziker W. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA. FEBS Lett. 1988 Oct 10;238(2):307–314. doi: 10.1016/0014-5793(88)80502-7. [DOI] [PubMed] [Google Scholar]
  20. Orning L., Gierse J. K., Fitzpatrick F. A. The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity. J Biol Chem. 1994 Apr 15;269(15):11269–11273. [PubMed] [Google Scholar]
  21. Orning L., Krivi G., Fitzpatrick F. A. Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes. J Biol Chem. 1991 Jan 25;266(3):1375–1378. [PubMed] [Google Scholar]
  22. Rawlings N. D., Barrett A. J. Evolutionary families of peptidases. Biochem J. 1993 Feb 15;290(Pt 1):205–218. doi: 10.1042/bj2900205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Vallee B. L., Auld D. S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry. 1990 Jun 19;29(24):5647–5659. doi: 10.1021/bi00476a001. [DOI] [PubMed] [Google Scholar]
  25. Vazeux G., Wang J., Corvol P., Llorens-Cortès C. Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A. J Biol Chem. 1996 Apr 12;271(15):9069–9074. doi: 10.1074/jbc.271.15.9069. [DOI] [PubMed] [Google Scholar]
  26. Wang J., Cooper M. D. Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1222–1226. doi: 10.1073/pnas.90.4.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wetterholm A., Medina J. F., Rådmark O., Shapiro R., Haeggström J. Z., Vallee B. L., Samuelsson B. Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9141–9145. doi: 10.1073/pnas.89.19.9141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wu Q., Lahti J. M., Air G. M., Burrows P. D., Cooper M. D. Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc Natl Acad Sci U S A. 1990 Feb;87(3):993–997. doi: 10.1073/pnas.87.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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