- Email: [email protected]
Peptidyl-Lys Metalloendopeptidase
1266
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
(Glu129, Asp143, and Tyr106) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis. J. Biol. Chem. 274(34), 2419524201. [14] McAuley, K.E., Jia-Xing, Y., Dodson, E.J., Lehmbeck, J., Ostergaard, P.R., Wilson, K.S. (2001). A quick solution: ab initio structure determination of a 19 kDa metalloproteinase using ACORN. Acta Crystallogr. D Biol. Crystallogr. 57(Pt 11), 15711578. [15] Matsumoto, K., Yamaguchi, M., Ichishima, E. (1994). Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, an 18 kDa metalloendopeptidase gene from Penicillium citrinum. Biochim. Biophys. Acta 1218, 469472.
[16] Ramesh, M.V., Sirakova, T.D., Kolattukudy, P.E. (1995). Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus. Gene 165, 121125. [17] Ichishima, E., Yamaguchi, M., Yano, H., Yamagata, Y., Hirano, K. (1991). Specificity of a new metalloproteinase form Penicillium citrinum. Agric. Biol. Chem. 55, 21912193. [18] Nonaka, T., Dohmae, N., Hashimoto, Y., Takio, K. (1997). Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biol. Chem. 272(48), 3003230039. [19] Su, N.W., Lee, M.H. (2001). Purification and characterization of a novel salt-tolerant protease from Aspergillus sp. FC-10, a soy sauce koji mold. J. Ind. Microbiol. Biotechnol. 26(4), 253258.
Hiroki Tatsumi Research & Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278, Japan. This article is reproduced from the previous edition with revisions made by the Editors. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00284-2
Chapter 284
Peptidyl-Lys Metalloendopeptidase DATABANKS MEROPS name: peptidyl-Lys metallopeptidase MEROPS classification: clan MA, subclan MA(M), family M35, peptidase M35.004 IUBMB: EC 3.4.24.20 (BRENDA) Tertiary structure: Available Species distribution: class Homobasidiomycetes Reference sequence from: Armillaria mellea (UniProt: Q9Y7F7)
Name and History The name peptidyl-Lys metalloendopeptidase is based on the quite selective substrate specificity (XaakLys) of the enzyme, i.e. at the N-terminal side of the lysine residue. The first enzyme with this specificity documented was Myxobacter AL-1 protease II (MyMEP) which was isolated from the culture fluid of Myxobacter strain AL-1 during CM-cellulose chromatography [1]. More recently, three enzymes, GfMEP (thermostable lysine-specific metalloendopeptidase), PoMEP (ProB) and AmMEP,
have been reported, extracted from the fruiting bodies of the basidiomycetes Grifola frondosa [24], Pleurotus ostreatus [5] and Armillaria mellea [6], respectively. A likely candidate of this group produced by a Gram-negative, facultatively anaerobic bacterium, Aeromonas hydrophila (AhMEP), has been reported, although no information is available other than the predicted amino acid sequence of the precursor protein [7].
Activity and Specificity From initial assays using the model substrates azocoll or azocasein [1] it was concluded that enzymes of this group have neutral to alkaline pH optima (AmMEP, 77.5; PoMEP, 8.5; MyMEP, 8.59; GfMEP, 9.5). Although PoMEP was reported to have optimum pH of 5.6 (with azocasein), re-examination with a larger group of synthetic peptide substrates revealed it to be 8.5. The acidic nature of azocasein may have caused it to give erroneous results [3]. Enzymes in this group are generally pH and temperature stable, and relatively resistant to denaturants.
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
PoMEP retains 96% activity after 5 h in 5 M urea at 37 C and pH 5 or 56% activity after 5 h in 2 M guanidine-HCl under the same conditions. An extensive evaluation of the GfMEP protease under typical proteomics conditions using a cellular lysate revealed that the enzyme is even extremely active under very harsh (denaturing) conditions (e.g. 6 M urea, 80% acetonitrile), and at temperatures as low as 4 C and up to 80 C [8]. GfMEP protease remains fully active in 4 M urea, and even at 6 M activity is estimated to be still at about 80%. One zinc atom per enzyme is an essential component; activities are inhibited by chelating agents such as EDTA and 1,10-phenanthroline. DTT also inhibits GfMEP (78% inhibition at 1 mM). Heavy metal ions inhibit MyMEP (Hg21, 36% inhibition at 0.1 mM), GfMEP (Hg21, 68% at 0.1 mM; Co21, 41% at 1 mM; Cu21, 31% at 1 mM) and PoMEP (at 1 mM, 90% inhibition with Hg21 or Ag1 and 70% inhibition with Cu21). Apoenzymes can be prepared by treatment of Gf- or PoMEP with 10 mM EDTA followed by dialysis. Activities are restored by the addition of divalent metal ions (GfMEP at 0.1 mM metal ion, .200% with Mn21, 140% with Zn21, 130% with Ca21 or 100% with Co21; PoMEP at 1 mM metal ion, 100% with Zn21, 80% with Co21 or 30% with Mn21). Inhibition by various amino acids and their derivatives has been reported. AmMEP is inhibited strongly by lysine (Ki 1 mM), n-propylamine (Ki 8 mM) and S-aminoethylcysteine (Ki 12.7 mM), but weakly by ornithine (Ki 140 mM) and arginine (Ki 170 mM). This inhibition spectrum is in accord with the specificity of the enzyme, suggesting specificity based on a rather rigid substrate pocket [16]. On the other hand, at a concentration of 5 mM
G A
LysN Trypsin 2.13 0.71 6.44 0.81
D, L-homoarginine (85%), agmatine (81%) and L-arginine (70%) inhibit GfMEP more strongly than L-lysine ethylester (66%) and L-lysine (42%) [4]. This result may indicate the presence of an acidic subsite in the substrate-binding site of GfMEP, in good agreement with the larger substrate recognition site described above. Activity is also severely hampered by guanidine hydrochloride and methanol [8]. Peptidyl-Lys metalloendopeptidases cleave preferentially acyl-lysine bonds in peptides and proteins. Cleavage of (XaakLys) by GfMEP is influenced by neighboring amino acids, but no single amino acid, not even proline, can completely prevent cleavage of the peptide bond next to the lysine residues [9]. Peptidyl-Lys metalloendopeptidases do not release terminal lysine residues. While S-aminoethylcysteine, a lysine homolog, is recognized as a substrate [10], neither arginine, ornithine nor D-lysine is recognized [3]. Simultaneous occurrence of two acidic residues at P1 and P3 or either Pro or Asp residue at the P20 position restricted cleavage [3,9,11]. While Gf- and PoMEP cleave polyarginine [4,5], no peptide bond other than XaakLys was cleaved in the selected synthetic peptides [3]. However, large-scale proteomics analysis did reveal that GfMEP purified from fruiting bodies also cleaves to some extent (XaakSer) and (XaakAla) bonds [8,12] (Figure 284.1). Kinetic parameters of Gf- and PoMEP were examined for various intramolecularly quenched fluorescent substrates [3]. While longer peptides were favored, a tripeptide DNP-TK-W was not cleaved. Co21 substituted PoMEP showed enhanced activity toward synthetic substrates although substrate preference was not altered [3]. GfMEP is
FIGURE 284.1 Specificity of cleavage of Gf MEP (LysN) and Trypsin as obtained from large-scale proteomics experiments. Percentage of total number of cleavages occurring at each of the amino acids residues.
70 LysN
P V L I M
0.89 2.25 2.32 1.28 0.83
0.64 1.11 1.62 1.16 0.58
60
F Y W S T
0.96 0.55 0.10 5.69 2.10
0.59 0.38 0.05 0.62 0.66
50
C N Q K H
0.08 1.71 1.17 67.54 0.18
0.11 0.63 0.74 54.11 0.45
R D E
1.23 1.41 1.15
33.66 0.55 0.82
Trypsin
50
30
20
10
0
G A P V L
I
1267
M F Y W S T C N Q K H R D E
1268
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
capable of, in contrast to trypsin, cleaving adjacent to mono- and dimethylated lysines, making it a potential candidate for targeted epigenetic analysis [8]. In contrast, GfMEP will not cleave adjacent to acetylated lysines.
Structural Chemistry The reported isoelectric points are pH 7.5 (GfMEP) and 8.4 (PoMEP). These enzymes contain one zinc atom per molecule (Am-, Gf- and PoMEP) as an essential component. Two enzymes, My- and AmMEP, are reported to be composed of 157 and 154 amino acids with minimum molecular masses of 16 600 and 16 650 Da, respectively [1]. Primary structures of the two mushroom enzymes have been determined [2] and nucleotide sequences of precursor proteins of AmMEP, GfMEP and AhMEP have been deposited in databases. They are homologous proteins of 167 (Gf MEP) and 168 (PoMEP and AmMEP) amino acid residues with 42.361.3% identity and molecular masses are calculated to be about 18 000 Da. They have two disulfide bonds and Thr42 of Gf MEP is partially mannosylated. They are found to share sequence homology (2126% sequence identity) with deuterolysins (Chapter 283) and with penicillolysin [13] (Chapter 282), GfMEP was found to be synthesized as a precursor protein with an 18 residues signal peptide and a 163 residues propeptide, which is similar to those of AmMEP, penicillolysin, and deuterolysins. Crystal structures of GfMEP (Figure 284.2) reveal a novel structure of five α-helices and four β-strands [14]. The structure is characterized by a long backbone helix (helix A) connecting a four-stranded β-sheet domain and a helical domain. Both domains provide sidewalls of the
FIGURE 284.2 Three-dimensional structure of GfMEP. The image was prepared from the Protein Data Bank entry (1G12) as described in the Introduction (p. li). The catalytic zinc ion is shown in CPK representation as a light gray sphere. The zinc ligands are shown in ball-and-stick representation: His117 and His121 in purple and Asp130 in pink. The catalytic Glu118 is also shown in ball-and-stick representation. Carbohydrates are shown as CPK spheres.
active site cleft and the active site helix (helix D) lines the bottom. Helix D contains two zinc ligands, His117 and His121, and the catalytic Glu118 as the components of the HEXXH motif. The loop D-E includes the strictly conserved GTXDXXYG segment in ‘aspzincins’, which contains Asp130 as the third zinc ligand (Figure 284.2). Asp130 makes a unique bifurcated interaction with the zinc ion. The fourth ligand is the catalytic water molecule, which also makes hydrogen bonds with both carboxyl oxygen atoms of Glu118. Unlike most MEPs, the coordination of the four ligands is not tetrahedral but pyramidal in GfMEP. The pyramidal base plane is formed by the ε-nitrogen atom of His117, both carboxyl oxygen atoms of Asp130, and the catalytic water and the ε-nitrogen atom of His121 is located at its vertex. Another water molecule acts as the fifth zinc ligand at the opposite side of the bipyramidal vertex His121. The δ-nitrogens of His117 and His121 interact with Asp154 and Thr128, respectively. In the surface electrostatic potential of GfMEP, a highly negative region is found at the bottom of the cleft surrounded by hydrophobic banks. The region is the putative S10 pocket to attract a positively charged amino group of Lys side chain specifically. Strict specificity of the peptidyl-lysine specific metalloendopeptidase is brought about from the exact distance of the catalytic zinc and the γ-carboxyl group of Glu157. Tyr133 located at the edge of the catalytic cleft was found in two different configurations (Figure 284.3). Only in monoclinic crystal, the fifth ligand water forms a hydrogen bond with the phenolic hydroxyl of Tyr133. In a transition state, a
FIGURE 284.3 GfMEP tetrahedral transition state model. The two structures (monoclinic and hexagonal crystal forms) are superimposed based on the zinc ligands. Residues related to catalytic reaction (His117, Glu118, His121, Asp130, Tyr133 and Glu157) in monoclinic crystal are presented with carbon atoms colored in green, oxygen in red and nitrogen in blue. Strand β3 is presented by thin stick model with carbon atoms colored in cyan. In hexagonal crystal, only Tyr133 is presented with carbon atoms in magenta. The modeled substrate (propionyl-AlaLys-Ala) is presented with carbon atoms in yellow. Putative hydrogen bonds and the salt-bridge between the substrate and GfMEP are drawn in broken lines. (Reproduced from Hori et al. [14] with permission)
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
scissile peptide bond would form a tetrahedral ‘waterattacked carbonyl group’ [15], and the locations of the two ligand water molecules are almost coincident with those of the two oxygen atoms of the water-attacked carbonyl group (Figure 284.3). The flexible Tyr133 would have two roles in the enzymatic function of GfMEP. One is a ‘proton donor’ to stabilize the reaction transition state and the other is a hydrophobic environment with Phe83 to accommodate the alkyl part of Lys side chain of substrates. A tyrosine residue of similar position, Tyr149, was reported to act as a general base during the catalysis in astacin (Chapter 188).
Preparation MyMEP [1] was purified from the culture medium using ammonium sulfate precipitation or column chromatography in 11.4% yield. AmMEP [16] was isolated from A. mellea caps in a yield of 35.1% (486-fold) and was homogeneous on PAGE at pH 4.3 and on SDS-PAGE. Gf- [4] and PoMEP [5] were purified 212- and 34.8-fold from homogenates of edible mushrooms, Grifola frondosa and Pleurotus ostreatus in yields of 24.7 and 8.3%, respectively, by conventional purification methods. Due to its potential applicability in proteomics the use of a recombinant peptidyl-Lys metalloendopeptidase enzyme has been reported, although details have not been published.
1269
major polysaccharides constituting the fungus cell wall, may provide an indication of the cellular localization and function of this enzyme.
Distinguishing Features Enzymes of this group have some unique features owing to their strict specificity, high heat stability, resistance to denaturing agents, and activity over a wide pH range. Recently, peptidyl-Lys metalloendopeptidases, in particular GfMEP, have been introduced as an alternative to the widely used trypsin in proteomics experiments [12,17,18]. Large-scale mass spectrometry analysis indicated that many of the peptides generated have their positive charges localized at the lysine N-terminus. Upon fragmentation by collision-induced dissociation (CID) in matrix assisted laser desorption ionization, or by electron transfer induced dissociation (ETD) upon electrospray ionization, peptides that contain just a single basic residue produce primarily N-terminal sequence ions, making interpretation of the fragmentation spectra rather straightforward [18,19]. In such an approach, proteins out of a cellular lysate are digested by GfMEP, and then ideally pre-fractionated by strong cation exchange, to enrich for sub-pools containing either single or multiple basis residues [20].
References Biological Aspects The enzymes are found exclusively in fruiting bodies of higher basidiomycetes, except for MyMEP. Although MyMEP is secreted into culture media, possibly to extract nutrients from extracellular proteins, the function of the mushroom enzymes internally is not clear. Content of the enzyme in the fruiting bodies is quite high, 0.53% of soluble proteins. It is not known yet whether the enzyme is found also in mycelia or secreted into culture medium (host tissue). AhMEP has a suggested involvement in infection. Unlike many serine proteinases, no intrinsic inhibitor nor inhibitory propeptide is likely to exist since the enzyme is fully active in the homogenate [5]. Gf MEP, but not PoMEP, binds to cellulose, curdlan (β-1,3-glucan) and chitin [4]. No activity could be detected in the breakthrough fraction when GfMEP was loaded onto a cellulose column at pH 5.0. Buffers containing 1.2 M KCl, 20 mM EDTA, 4 M urea or 0.5 M cellobiose failed to elute bound enzyme. This enzyme can be eluted quantitatively with a pH 8.5 buffer containing 1.2 M KCl. GfMEP is fully active in the presence of cellulose. However, GfMEP passes through a Sephadex G-25 column at pH 5.0, indicating that the enzyme lacks affinity for dextran, α1-1,6-linked glucose polymer. Its strong interaction with both β-1,3-glucan and chitin,
[1] Wingard, M., Matsueda, G., Wolfe, R.S. (1972). Myxobacter AL-1 protease II: specific peptide bond cleavage on the amino side of lysine. J. Bacteriol. 112, 940949. [2] Nonaka, T., Dohmae, N., Hashimoto, Y., Takio, K. (1997). Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biol. Chem. 272, 3003230039. [3] Nonaka, T., Hashimoto, Y., Takio, K. (1998). Kinetic characterization of lysine-specific metalloendopeptidases from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biochem. 124, 157162. [4] Nonaka, T., Ishikawa, H., Tsumuraya, Y., Hashimoto, Y., Dohmae, N. (1995). Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa. J. Biochem. 118, 10141020. [5] Dohmae, N., Hayashi, K., Miki, K., Tsumuraya, Y., Hashimoto, Y. (1995). Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies. Biosci. Biotechnol. Biochem. 59, 20742080. [6] Healy, V., O’Connell, J., McCarthy, T.V., Doonan, S. (1999). The lysine-specific proteinase from Armillaria mellea is a member of a novel class of metalloendopeptidases located in basidiomycetes. Biochem. Biophys. Res. Commun. 262(1), 6063. [7] Chang, T.M., Liu, C.C., Chang, M.C. (1997). Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila. Gene 199, 225229. [8] Taouatas, N., Heck, A.J., Mohammed, S. (2010). Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. J. Proteome Res. 9, 42824288.
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[9] Raijmakers, R., Neerincx, P., Mohammed, S., Heck, A.J. (2010). Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chem. Commun. (Camb.) 46, 88278829. [10] Doonan, S., Fahmy, H.M. (1975). Specific enzymic cleavage of polypeptides at cysteine residues. Eur. J. Biochem. 56, 421426. [11] Shipolini, R.A., Callewaert, G.L., Cottrell, R.C., Vernon, C.A. (1974). The amino-acid sequence and carbohydrate content of phospholipase A2 from bee venom. Eur. J. Biochem. 48, 465476. [12] Hohmann, L., Sherwood, C., Eastham, A., Peterson, A., Eng, J.K., Eddes, J.S., Shteynberg, D., Martin, D.B. (2009). Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N. J. Proteome Res. 8, 14151422. [13] Matsumoto, K., Yamaguchi, M., Ichishima, E. (1994). Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, and 18 kDa metalloendopeptidase gene from Penicillium citrinum. Biochim. Biophys. Acta 1218, 469472. [14] Hori, T., Kumasaka, T., Yamamoto, M., Nonaka, N., Tanaka, N., Hashimoto, Y., Ueki, U., Takio, K. (2001). Structure of a new ‘aspzincin’ metalloendopeptidase from Grifola frondosa: implications for the catalytic mechanism and substrate specificity based on several different crystal forms. Acta Crystallogr. D Biol. Crystallogr. 57, 361368. [15] Holden, H.M., Tronrud, D.E., Monzingo, A.F., Weaver, L.H., Matthews, B.W. (1987). Slow- and fast-binding inhibitors of
[16]
[17]
[18]
[19]
[20]
thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry 26, 85428553. Lewis, W.G., Basford, J.M., Walton, P.L. (1978). Specificity and inhibition studies of Armillaria mellea protease. Biochim. Biophys. Acta 522, 551560. Aye, T.T., Scholten, A., Taouatas, N., Varro, A., Van Veen, T.A., Vos, M.A., Heck, A.J. (2010). Proteome-wide protein concentrations in the human heart. Mol. Biosyst. 6, 19171927. Taouatas, N., Drugan, M.M., Heck, A.J., Mohammed, S. (2008). Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase. Nat. Methods 5, 405407. Boersema, P.J., Taouatas, N., Altelaar, A.F., Gouw, J.W., Ross, P.L., Pappin, D.J., Heck, A.J., Mohammed, S. (2009). Straightforward and de novo peptide sequencing by MALDI-MS/ MS using a Lys-N metalloendopeptidase. Mol. Cell. Proteomics 8, 650660. Taouatas, N., Altelaar, A.F., Drugan, M.M., Helbig, A.O., Mohammed, S., Heck, A.J. (2009). Strong cation exchange-based fractionation of Lys-N-generated peptides facilitates the targeted analysis of post-translational modifications. Mol. Cell. Proteomics 8, 190200.
Albert J.R. Heck Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Email: [email protected] This article is a revision of the previous edition article by Koji Takio, Volume 1, pp. 788792, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00285-4
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
(Glu129, Asp143, and Tyr106) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis. J. Biol. Chem. 274(34), 2419524201. [14] McAuley, K.E., Jia-Xing, Y., Dodson, E.J., Lehmbeck, J., Ostergaard, P.R., Wilson, K.S. (2001). A quick solution: ab initio structure determination of a 19 kDa metalloproteinase using ACORN. Acta Crystallogr. D Biol. Crystallogr. 57(Pt 11), 15711578. [15] Matsumoto, K., Yamaguchi, M., Ichishima, E. (1994). Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, an 18 kDa metalloendopeptidase gene from Penicillium citrinum. Biochim. Biophys. Acta 1218, 469472.
[16] Ramesh, M.V., Sirakova, T.D., Kolattukudy, P.E. (1995). Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus. Gene 165, 121125. [17] Ichishima, E., Yamaguchi, M., Yano, H., Yamagata, Y., Hirano, K. (1991). Specificity of a new metalloproteinase form Penicillium citrinum. Agric. Biol. Chem. 55, 21912193. [18] Nonaka, T., Dohmae, N., Hashimoto, Y., Takio, K. (1997). Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biol. Chem. 272(48), 3003230039. [19] Su, N.W., Lee, M.H. (2001). Purification and characterization of a novel salt-tolerant protease from Aspergillus sp. FC-10, a soy sauce koji mold. J. Ind. Microbiol. Biotechnol. 26(4), 253258.
Hiroki Tatsumi Research & Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278, Japan. This article is reproduced from the previous edition with revisions made by the Editors. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00284-2
Chapter 284
Peptidyl-Lys Metalloendopeptidase DATABANKS MEROPS name: peptidyl-Lys metallopeptidase MEROPS classification: clan MA, subclan MA(M), family M35, peptidase M35.004 IUBMB: EC 3.4.24.20 (BRENDA) Tertiary structure: Available Species distribution: class Homobasidiomycetes Reference sequence from: Armillaria mellea (UniProt: Q9Y7F7)
Name and History The name peptidyl-Lys metalloendopeptidase is based on the quite selective substrate specificity (XaakLys) of the enzyme, i.e. at the N-terminal side of the lysine residue. The first enzyme with this specificity documented was Myxobacter AL-1 protease II (MyMEP) which was isolated from the culture fluid of Myxobacter strain AL-1 during CM-cellulose chromatography [1]. More recently, three enzymes, GfMEP (thermostable lysine-specific metalloendopeptidase), PoMEP (ProB) and AmMEP,
have been reported, extracted from the fruiting bodies of the basidiomycetes Grifola frondosa [24], Pleurotus ostreatus [5] and Armillaria mellea [6], respectively. A likely candidate of this group produced by a Gram-negative, facultatively anaerobic bacterium, Aeromonas hydrophila (AhMEP), has been reported, although no information is available other than the predicted amino acid sequence of the precursor protein [7].
Activity and Specificity From initial assays using the model substrates azocoll or azocasein [1] it was concluded that enzymes of this group have neutral to alkaline pH optima (AmMEP, 77.5; PoMEP, 8.5; MyMEP, 8.59; GfMEP, 9.5). Although PoMEP was reported to have optimum pH of 5.6 (with azocasein), re-examination with a larger group of synthetic peptide substrates revealed it to be 8.5. The acidic nature of azocasein may have caused it to give erroneous results [3]. Enzymes in this group are generally pH and temperature stable, and relatively resistant to denaturants.
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
PoMEP retains 96% activity after 5 h in 5 M urea at 37 C and pH 5 or 56% activity after 5 h in 2 M guanidine-HCl under the same conditions. An extensive evaluation of the GfMEP protease under typical proteomics conditions using a cellular lysate revealed that the enzyme is even extremely active under very harsh (denaturing) conditions (e.g. 6 M urea, 80% acetonitrile), and at temperatures as low as 4 C and up to 80 C [8]. GfMEP protease remains fully active in 4 M urea, and even at 6 M activity is estimated to be still at about 80%. One zinc atom per enzyme is an essential component; activities are inhibited by chelating agents such as EDTA and 1,10-phenanthroline. DTT also inhibits GfMEP (78% inhibition at 1 mM). Heavy metal ions inhibit MyMEP (Hg21, 36% inhibition at 0.1 mM), GfMEP (Hg21, 68% at 0.1 mM; Co21, 41% at 1 mM; Cu21, 31% at 1 mM) and PoMEP (at 1 mM, 90% inhibition with Hg21 or Ag1 and 70% inhibition with Cu21). Apoenzymes can be prepared by treatment of Gf- or PoMEP with 10 mM EDTA followed by dialysis. Activities are restored by the addition of divalent metal ions (GfMEP at 0.1 mM metal ion, .200% with Mn21, 140% with Zn21, 130% with Ca21 or 100% with Co21; PoMEP at 1 mM metal ion, 100% with Zn21, 80% with Co21 or 30% with Mn21). Inhibition by various amino acids and their derivatives has been reported. AmMEP is inhibited strongly by lysine (Ki 1 mM), n-propylamine (Ki 8 mM) and S-aminoethylcysteine (Ki 12.7 mM), but weakly by ornithine (Ki 140 mM) and arginine (Ki 170 mM). This inhibition spectrum is in accord with the specificity of the enzyme, suggesting specificity based on a rather rigid substrate pocket [16]. On the other hand, at a concentration of 5 mM
G A
LysN Trypsin 2.13 0.71 6.44 0.81
D, L-homoarginine (85%), agmatine (81%) and L-arginine (70%) inhibit GfMEP more strongly than L-lysine ethylester (66%) and L-lysine (42%) [4]. This result may indicate the presence of an acidic subsite in the substrate-binding site of GfMEP, in good agreement with the larger substrate recognition site described above. Activity is also severely hampered by guanidine hydrochloride and methanol [8]. Peptidyl-Lys metalloendopeptidases cleave preferentially acyl-lysine bonds in peptides and proteins. Cleavage of (XaakLys) by GfMEP is influenced by neighboring amino acids, but no single amino acid, not even proline, can completely prevent cleavage of the peptide bond next to the lysine residues [9]. Peptidyl-Lys metalloendopeptidases do not release terminal lysine residues. While S-aminoethylcysteine, a lysine homolog, is recognized as a substrate [10], neither arginine, ornithine nor D-lysine is recognized [3]. Simultaneous occurrence of two acidic residues at P1 and P3 or either Pro or Asp residue at the P20 position restricted cleavage [3,9,11]. While Gf- and PoMEP cleave polyarginine [4,5], no peptide bond other than XaakLys was cleaved in the selected synthetic peptides [3]. However, large-scale proteomics analysis did reveal that GfMEP purified from fruiting bodies also cleaves to some extent (XaakSer) and (XaakAla) bonds [8,12] (Figure 284.1). Kinetic parameters of Gf- and PoMEP were examined for various intramolecularly quenched fluorescent substrates [3]. While longer peptides were favored, a tripeptide DNP-TK-W was not cleaved. Co21 substituted PoMEP showed enhanced activity toward synthetic substrates although substrate preference was not altered [3]. GfMEP is
FIGURE 284.1 Specificity of cleavage of Gf MEP (LysN) and Trypsin as obtained from large-scale proteomics experiments. Percentage of total number of cleavages occurring at each of the amino acids residues.
70 LysN
P V L I M
0.89 2.25 2.32 1.28 0.83
0.64 1.11 1.62 1.16 0.58
60
F Y W S T
0.96 0.55 0.10 5.69 2.10
0.59 0.38 0.05 0.62 0.66
50
C N Q K H
0.08 1.71 1.17 67.54 0.18
0.11 0.63 0.74 54.11 0.45
R D E
1.23 1.41 1.15
33.66 0.55 0.82
Trypsin
50
30
20
10
0
G A P V L
I
1267
M F Y W S T C N Q K H R D E
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Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
capable of, in contrast to trypsin, cleaving adjacent to mono- and dimethylated lysines, making it a potential candidate for targeted epigenetic analysis [8]. In contrast, GfMEP will not cleave adjacent to acetylated lysines.
Structural Chemistry The reported isoelectric points are pH 7.5 (GfMEP) and 8.4 (PoMEP). These enzymes contain one zinc atom per molecule (Am-, Gf- and PoMEP) as an essential component. Two enzymes, My- and AmMEP, are reported to be composed of 157 and 154 amino acids with minimum molecular masses of 16 600 and 16 650 Da, respectively [1]. Primary structures of the two mushroom enzymes have been determined [2] and nucleotide sequences of precursor proteins of AmMEP, GfMEP and AhMEP have been deposited in databases. They are homologous proteins of 167 (Gf MEP) and 168 (PoMEP and AmMEP) amino acid residues with 42.361.3% identity and molecular masses are calculated to be about 18 000 Da. They have two disulfide bonds and Thr42 of Gf MEP is partially mannosylated. They are found to share sequence homology (2126% sequence identity) with deuterolysins (Chapter 283) and with penicillolysin [13] (Chapter 282), GfMEP was found to be synthesized as a precursor protein with an 18 residues signal peptide and a 163 residues propeptide, which is similar to those of AmMEP, penicillolysin, and deuterolysins. Crystal structures of GfMEP (Figure 284.2) reveal a novel structure of five α-helices and four β-strands [14]. The structure is characterized by a long backbone helix (helix A) connecting a four-stranded β-sheet domain and a helical domain. Both domains provide sidewalls of the
FIGURE 284.2 Three-dimensional structure of GfMEP. The image was prepared from the Protein Data Bank entry (1G12) as described in the Introduction (p. li). The catalytic zinc ion is shown in CPK representation as a light gray sphere. The zinc ligands are shown in ball-and-stick representation: His117 and His121 in purple and Asp130 in pink. The catalytic Glu118 is also shown in ball-and-stick representation. Carbohydrates are shown as CPK spheres.
active site cleft and the active site helix (helix D) lines the bottom. Helix D contains two zinc ligands, His117 and His121, and the catalytic Glu118 as the components of the HEXXH motif. The loop D-E includes the strictly conserved GTXDXXYG segment in ‘aspzincins’, which contains Asp130 as the third zinc ligand (Figure 284.2). Asp130 makes a unique bifurcated interaction with the zinc ion. The fourth ligand is the catalytic water molecule, which also makes hydrogen bonds with both carboxyl oxygen atoms of Glu118. Unlike most MEPs, the coordination of the four ligands is not tetrahedral but pyramidal in GfMEP. The pyramidal base plane is formed by the ε-nitrogen atom of His117, both carboxyl oxygen atoms of Asp130, and the catalytic water and the ε-nitrogen atom of His121 is located at its vertex. Another water molecule acts as the fifth zinc ligand at the opposite side of the bipyramidal vertex His121. The δ-nitrogens of His117 and His121 interact with Asp154 and Thr128, respectively. In the surface electrostatic potential of GfMEP, a highly negative region is found at the bottom of the cleft surrounded by hydrophobic banks. The region is the putative S10 pocket to attract a positively charged amino group of Lys side chain specifically. Strict specificity of the peptidyl-lysine specific metalloendopeptidase is brought about from the exact distance of the catalytic zinc and the γ-carboxyl group of Glu157. Tyr133 located at the edge of the catalytic cleft was found in two different configurations (Figure 284.3). Only in monoclinic crystal, the fifth ligand water forms a hydrogen bond with the phenolic hydroxyl of Tyr133. In a transition state, a
FIGURE 284.3 GfMEP tetrahedral transition state model. The two structures (monoclinic and hexagonal crystal forms) are superimposed based on the zinc ligands. Residues related to catalytic reaction (His117, Glu118, His121, Asp130, Tyr133 and Glu157) in monoclinic crystal are presented with carbon atoms colored in green, oxygen in red and nitrogen in blue. Strand β3 is presented by thin stick model with carbon atoms colored in cyan. In hexagonal crystal, only Tyr133 is presented with carbon atoms in magenta. The modeled substrate (propionyl-AlaLys-Ala) is presented with carbon atoms in yellow. Putative hydrogen bonds and the salt-bridge between the substrate and GfMEP are drawn in broken lines. (Reproduced from Hori et al. [14] with permission)
Clan MA (E) M35 | 284. Peptidyl-Lys Metalloendopeptidase
scissile peptide bond would form a tetrahedral ‘waterattacked carbonyl group’ [15], and the locations of the two ligand water molecules are almost coincident with those of the two oxygen atoms of the water-attacked carbonyl group (Figure 284.3). The flexible Tyr133 would have two roles in the enzymatic function of GfMEP. One is a ‘proton donor’ to stabilize the reaction transition state and the other is a hydrophobic environment with Phe83 to accommodate the alkyl part of Lys side chain of substrates. A tyrosine residue of similar position, Tyr149, was reported to act as a general base during the catalysis in astacin (Chapter 188).
Preparation MyMEP [1] was purified from the culture medium using ammonium sulfate precipitation or column chromatography in 11.4% yield. AmMEP [16] was isolated from A. mellea caps in a yield of 35.1% (486-fold) and was homogeneous on PAGE at pH 4.3 and on SDS-PAGE. Gf- [4] and PoMEP [5] were purified 212- and 34.8-fold from homogenates of edible mushrooms, Grifola frondosa and Pleurotus ostreatus in yields of 24.7 and 8.3%, respectively, by conventional purification methods. Due to its potential applicability in proteomics the use of a recombinant peptidyl-Lys metalloendopeptidase enzyme has been reported, although details have not been published.
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major polysaccharides constituting the fungus cell wall, may provide an indication of the cellular localization and function of this enzyme.
Distinguishing Features Enzymes of this group have some unique features owing to their strict specificity, high heat stability, resistance to denaturing agents, and activity over a wide pH range. Recently, peptidyl-Lys metalloendopeptidases, in particular GfMEP, have been introduced as an alternative to the widely used trypsin in proteomics experiments [12,17,18]. Large-scale mass spectrometry analysis indicated that many of the peptides generated have their positive charges localized at the lysine N-terminus. Upon fragmentation by collision-induced dissociation (CID) in matrix assisted laser desorption ionization, or by electron transfer induced dissociation (ETD) upon electrospray ionization, peptides that contain just a single basic residue produce primarily N-terminal sequence ions, making interpretation of the fragmentation spectra rather straightforward [18,19]. In such an approach, proteins out of a cellular lysate are digested by GfMEP, and then ideally pre-fractionated by strong cation exchange, to enrich for sub-pools containing either single or multiple basis residues [20].
References Biological Aspects The enzymes are found exclusively in fruiting bodies of higher basidiomycetes, except for MyMEP. Although MyMEP is secreted into culture media, possibly to extract nutrients from extracellular proteins, the function of the mushroom enzymes internally is not clear. Content of the enzyme in the fruiting bodies is quite high, 0.53% of soluble proteins. It is not known yet whether the enzyme is found also in mycelia or secreted into culture medium (host tissue). AhMEP has a suggested involvement in infection. Unlike many serine proteinases, no intrinsic inhibitor nor inhibitory propeptide is likely to exist since the enzyme is fully active in the homogenate [5]. Gf MEP, but not PoMEP, binds to cellulose, curdlan (β-1,3-glucan) and chitin [4]. No activity could be detected in the breakthrough fraction when GfMEP was loaded onto a cellulose column at pH 5.0. Buffers containing 1.2 M KCl, 20 mM EDTA, 4 M urea or 0.5 M cellobiose failed to elute bound enzyme. This enzyme can be eluted quantitatively with a pH 8.5 buffer containing 1.2 M KCl. GfMEP is fully active in the presence of cellulose. However, GfMEP passes through a Sephadex G-25 column at pH 5.0, indicating that the enzyme lacks affinity for dextran, α1-1,6-linked glucose polymer. Its strong interaction with both β-1,3-glucan and chitin,
[1] Wingard, M., Matsueda, G., Wolfe, R.S. (1972). Myxobacter AL-1 protease II: specific peptide bond cleavage on the amino side of lysine. J. Bacteriol. 112, 940949. [2] Nonaka, T., Dohmae, N., Hashimoto, Y., Takio, K. (1997). Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biol. Chem. 272, 3003230039. [3] Nonaka, T., Hashimoto, Y., Takio, K. (1998). Kinetic characterization of lysine-specific metalloendopeptidases from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J. Biochem. 124, 157162. [4] Nonaka, T., Ishikawa, H., Tsumuraya, Y., Hashimoto, Y., Dohmae, N. (1995). Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa. J. Biochem. 118, 10141020. [5] Dohmae, N., Hayashi, K., Miki, K., Tsumuraya, Y., Hashimoto, Y. (1995). Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies. Biosci. Biotechnol. Biochem. 59, 20742080. [6] Healy, V., O’Connell, J., McCarthy, T.V., Doonan, S. (1999). The lysine-specific proteinase from Armillaria mellea is a member of a novel class of metalloendopeptidases located in basidiomycetes. Biochem. Biophys. Res. Commun. 262(1), 6063. [7] Chang, T.M., Liu, C.C., Chang, M.C. (1997). Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila. Gene 199, 225229. [8] Taouatas, N., Heck, A.J., Mohammed, S. (2010). Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. J. Proteome Res. 9, 42824288.
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[9] Raijmakers, R., Neerincx, P., Mohammed, S., Heck, A.J. (2010). Cleavage specificities of the brother and sister proteases Lys-C and Lys-N. Chem. Commun. (Camb.) 46, 88278829. [10] Doonan, S., Fahmy, H.M. (1975). Specific enzymic cleavage of polypeptides at cysteine residues. Eur. J. Biochem. 56, 421426. [11] Shipolini, R.A., Callewaert, G.L., Cottrell, R.C., Vernon, C.A. (1974). The amino-acid sequence and carbohydrate content of phospholipase A2 from bee venom. Eur. J. Biochem. 48, 465476. [12] Hohmann, L., Sherwood, C., Eastham, A., Peterson, A., Eng, J.K., Eddes, J.S., Shteynberg, D., Martin, D.B. (2009). Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N. J. Proteome Res. 8, 14151422. [13] Matsumoto, K., Yamaguchi, M., Ichishima, E. (1994). Molecular cloning and nucleotide sequence of the complementary DNA for penicillolysin gene, plnC, and 18 kDa metalloendopeptidase gene from Penicillium citrinum. Biochim. Biophys. Acta 1218, 469472. [14] Hori, T., Kumasaka, T., Yamamoto, M., Nonaka, N., Tanaka, N., Hashimoto, Y., Ueki, U., Takio, K. (2001). Structure of a new ‘aspzincin’ metalloendopeptidase from Grifola frondosa: implications for the catalytic mechanism and substrate specificity based on several different crystal forms. Acta Crystallogr. D Biol. Crystallogr. 57, 361368. [15] Holden, H.M., Tronrud, D.E., Monzingo, A.F., Weaver, L.H., Matthews, B.W. (1987). Slow- and fast-binding inhibitors of
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thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues. Biochemistry 26, 85428553. Lewis, W.G., Basford, J.M., Walton, P.L. (1978). Specificity and inhibition studies of Armillaria mellea protease. Biochim. Biophys. Acta 522, 551560. Aye, T.T., Scholten, A., Taouatas, N., Varro, A., Van Veen, T.A., Vos, M.A., Heck, A.J. (2010). Proteome-wide protein concentrations in the human heart. Mol. Biosyst. 6, 19171927. Taouatas, N., Drugan, M.M., Heck, A.J., Mohammed, S. (2008). Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase. Nat. Methods 5, 405407. Boersema, P.J., Taouatas, N., Altelaar, A.F., Gouw, J.W., Ross, P.L., Pappin, D.J., Heck, A.J., Mohammed, S. (2009). Straightforward and de novo peptide sequencing by MALDI-MS/ MS using a Lys-N metalloendopeptidase. Mol. Cell. Proteomics 8, 650660. Taouatas, N., Altelaar, A.F., Drugan, M.M., Helbig, A.O., Mohammed, S., Heck, A.J. (2009). Strong cation exchange-based fractionation of Lys-N-generated peptides facilitates the targeted analysis of post-translational modifications. Mol. Cell. Proteomics 8, 190200.
Albert J.R. Heck Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Email: [email protected] This article is a revision of the previous edition article by Koji Takio, Volume 1, pp. 788792, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00285-4