Aspzincin, a Family of Metalloendopeptidases with a New Zinc-binding Motif

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His128 and His132 with Arg, of Glu129 with Gln or Asp, of Asp143 with Asn or Glu, of Asp164 with Asn, and of Tyr106 with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu129, Asp143, and Tyr106 are catalytically crucial residues in the enzyme. Glu129 may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn2+ metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the “HEXXH + D” motif and an aspartic acid as the third zinc ligand.

Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu 129 , Asp 143 , and Tyr 106 are catalytically crucial residues in the enzyme. Glu 129 may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn 2؉ metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH ؉ D" motif and an aspartic acid as the third zinc ligand.
Metalloendopeptidases are physiologically important proteinases for processing proteins in eukaryotes and prokaryotes and are one of the best known classes of proteolytic enzymes (1,2). From recent data, Hooper (3) attempted to present a scheme based on the zinc-binding site, and this has been extended to classify zinc metalloproteases into distinct families. There are gluzincins, metzincins, inverzincins, the carboxypeptidase family, and the DD-carboxypeptidase family. Rawlings and Barrett (4) divided the families of metallopeptidases into five groups based on the zinc-binding motif: "HEXXH ϩ E", "HEXXH ϩ H", "HEXXH ϩ ?", heterogeneous other than HEXXH, and unknown.
The molds Aspergillus oryzae and Aspergillus sojae are of great practical importance in Japanese fermentation industries and enzyme technologies (5). In the fermented vegetable protein, soy sauce, the cooked soybeans are mixed with equal amounts of roasted wheat and then inoculated with a pure cultured starter of A. sojae or A. oryzae, which is called "koji starter" or "seed mold" (6). Sekine et al. (7) previously reported that both neutral proteinases I and II were found to have an effect equal to that of alkaline serine proteinase (8,9) on the hydrolysis and liquefaction of soybean protein. Neutral proteinase II is now designated as deuterolysin (EC 3.4.24.39) according to the Enzyme Nomenclature Commission (10). The metalloproteinases (neutral proteinases I and II) from A. sojae were characterized by Sekine (11)(12)(13)(14)(15).
Neutral proteinase I contains 1 g atom of zinc and 2 g atoms of calcium per mol of enzyme, and the zinc is essential for activity (13). Neutral proteinase I with a molecular mass of 41,700 Da has enzymatic properties similar to those of the neutral proteinase from Bacillus thermoproteolyticus, thermolysin (EC 3.4.24.27) (15). In the digestion of the oxidized insulin B-chain with neutral proteinase I, the cleavage sites produced by the enzyme are very similar to those of the other neutral proteinases.
Deuterolysin from A. sojae possesses a preference for basic proteins as substrates, showing high activities on the basic nuclear proteins histone, protamine, and salmine, but very low activities on milk casein, hemoglobin, albumin, and gelatin (12,14). Deuterolysin is extremely stable at 100°C, but relatively unstable around 75°C (16). Elucidation of the thermal stability at 100°C of the deuterolysin from A. oryzae has also been reported and discussed. It contains 1 g atom of zinc and 2 g atoms of calcium per mol with a molecular mass of 19,800 Da (13) and includes three disulfide bonds (16). The cloning and expression in yeast cells of a cDNA clone (S53810) encoding deuterolysin from A. oryzae were reported by Tatsumi et al. (17).
The elucidation of substrate specificity with an oxidized insulin B-chain (18) and bioactive oligopeptides (19) led to the discovery of penicillolysin as an enzyme thought to be a new 18-kDa metalloendopeptidase from Penicillium citrinum, having a distinct mode of action and a specificity unique from those of other metalloendopeptidases (2, 3, 20 -22). Penicillolysin also possesses a preference for basic proteins as substrates, showing high activities on basic nuclear proteins such as histone, protamine, and salmine, but very low activities on milk casein, hemoglobin, albumin, and gelatin (19); however, the enzyme has no heat stability above 60°C. Penicillolysin contains 1 g atom of zinc/mol of enzyme and three disulfide bonds. The enzyme is a single-chain protein of 177 amino acid residues with a molecular mass of 18,529 Da and pI 9.6. Cloning of a cDNA clone (D25535) encoding penicillolysin from P. citrinum was carried out by Matsumoto et al. (23). Penicillolysin shows 68% sequence identity to deuterolysin from A. oryzae. We previously assumed that His 128 , His 132 , and Glu 65 of penicillolysin (23) corresponded to zinc ligands in thermolysin (24) and the neutral proteinases (2, 3).
In this paper, the possibility of a zinc-binding role for Glu 65 in deuterolysin was ruled out because the mutant E65Q still had catalytic activity. The predicted amino acid sequence, including aspartic acid and glutamic acid, of deuterolysin (17) has strong similarity to other members of the deuterolysin family: MEP20 from Aspergillus flavus (25) and Aspergillus fumigatus (26), metalloendopeptidases from Grifola frondosa and Pleurotus ostreatus (27), and penicillolysin (23). This finding may indicate that enzymes in the deuterolysin family are coded for by evolutionarily related genes at the enzymatic level. We hypothesized that Asp 143 or Asp 164 in the highly conserved region of the C terminus may be the third ligand of deuterolysin.
We describe here site-directed mutagenesis studies of deuterolysin from A. oryzae for active-site determination. We found a new zinc-binding motif, aspzincin, defined as the "HEXXH ϩ D" motif with an aspartic acid as the third zinc ligand.
Isolation of mRNA from A. oryzae-A. oryzae IFO 4251 was cultured in 50 ml of wheat bran medium (1.5% defatted soybean peptone, 1% potassium phosphate, and 0.02% zinc chloride dissolved in wheat bran extract, pH 6.8, at 37°C for 80 h), and the culture was centrifuged to obtain 2 g of wet cells. Total RNA was isolated and purified by the method of Chomczynski and Sacchi (28). Poly(A) ϩ RNA was isolated using an Oligotex-dT30 (Takara Shuzo).
Slight Variation in the Sequence of Deuterolysin-The amino acid sequence of deuterolysin from A. oryzae IFO 4251 was identical to that (cDNA of neutral proteinase II) reported by Tatsumi et al. (17) except for the substitution of three codons. The different codons were CTC (for nucleotides 48 -50; Leu for amino acid 6 in the preregion) replaced by ATC (Ile), ACT (for nucleotides 72-74; Thr for amino acid 14 in the preregion) replaced by GCT (Ala), and CTT (for nucleotides 894 -896; Leu for amino acid 113 in the mature region) replaced by TTG (Leu). There was no remarkable change in the amino acid sequences of the two mature enzymes deduced.
Construction of Expression Plasmids in E. coli Cells-The expression plasmid for prodeuterolysin, pETDLN, was constructed by inserting the deuterolysin cDNA fragment with the T7 promoter/lac operator and T7 terminator and thioredoxin fusion gene (31) into pET32a (Novagen). The cDNA was modified by polymerase chain reaction to remove the signal sequence and to introduce a start codon at the N terminus of prodeuterolysin. The sense primer 5Ј-TTGaaTTCCACCGCTGccatg-GCGCCAACCGCT-3Ј and antisense primer 5Ј-GAGGgATCcTTCA-CATTTAGCACTTGAGCTC-3Ј introduced EcoRI, NcoI, and BamHI sites at the 5Ј-end, N terminus, and 3Ј-end, respectively. Mutation sites with the original sequence of dlnO are indicated in lowercase letters. A Met start codon was created by the NcoI site at the N terminus of prodeuterolysin. The amplified 1.0-kilobase pair cDNA fragment was digested with EcoRI and BamHI and cloned into pUC119 to generate pUCDLN(EB). The amplified cDNA was confirmed to lack the undesired mutation by sequencing. The NcoI-BamHI fragment from the pUCDLN(EB) was subcloned into the pET32a vector to generate pET-DLN ( Fig. 1).
Expression of Prodeuterolysin in E. coli -E. coli BL21(DE3) cells transformed with pETDLN were cultured at 37°C in a 5-liter jar fermentor containing 2 liters of 4-fold concentrated LB medium containing 50 mg/ml ampicillin, 10 mM calcium chloride, and 0.25% (v/v) antifoam DB110N emulsion until A 600 nm reached 7; then isopropyl-␤-D-thiogalactopyranoside was added to a final concentration of 1 mM, and incubation was continued for 3 h (aeration and agitation rate was 3 liters/min and 300 rpm, respectively). The cells were harvested by centrifugation at 15,000 ϫ g for 20 min, resuspended in 200 ml of 10 mM Tris-HCl buffer, pH 8.0, and frozen at Ϫ80°C. sonication. The cell homogenate was centrifuged at 20,000 ϫ g for 10 min. The supernatant was recentrifuged, and the resulting supernatant was used as crude prodeuterolysin preparation. The crude proenzyme solution was loaded on a RESOURCE Q column (0.64 ϫ 3 cm, Amersham Pharmacia Biotech) and eluted with 5 mM sodium phosphate, pH 7.0, with a linear gradient of 0 -0.5 M NaCl. The fractions containing prodeuterolysin were pooled. The pooled fraction was loaded on a Hi-Load Superdex 75 column (1.6 ϫ 60 cm, Amersham Pharmacia Biotech) and eluted with 5 mM sodium phosphate buffer, pH 7.0, containing 0.3 M NaCl. The fractions containing prodeuterolysin were dialyzed against 5 mM sodium phosphate buffer, pH 7.0, and used as a purified prodeuterolysin preparation.
Conversion of Prodeuterolysin to the Mature Form-The purified proenzyme fraction was incubated at 37°C for 30 min with an equal amount of trypsin (mol/mol, Merck). After the addition of L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone hydrochloride at 100 M as a trypsin inhibitor, the reaction mixture was loaded on a HiLoad 16/60 Superdex 75 column (1.6 ϫ 60 cm, Amersham Pharmacia Biotech) and eluted with 5 mM sodium phosphate buffer, pH 7.0, containing 0.3 M NaCl. The fractions containing deuterolysin were pooled. After dialysis against 5 mM sodium phosphate buffer, pH 7.0, the pooled fraction was loaded on two HiTrap Q anion-exchange columns (0.77 ϫ 2.5 cm, Amersham Pharmacia Biotech) and eluted with 5 mM sodium phosphate, pH 7.0, with a linear gradient of 0 -0.5 M NaCl. The fractions containing activated deuterolysin were pooled. Maturation from prodeuterolysin to active deuterolysin was also observed with the addition of 5 mM Zn 2ϩ .
Proteolytic Activity Assay-Proteolytic activities with salmon protamine sulfate (salmine) were assayed at pH 7.0 and 30°C. Fifty l of sample and 400 l of 100 mM sodium phosphate buffer, pH 7.0, were mixed and preincubated for 5 min, and then 150 l of 2% (w/v) salmon protamine sulfate (previously denatured at 100°C for 30 min in 100 mM sodium phosphate buffer, pH 7.0) was added and incubated for various periods. Six-hundred ml of 12.5% (w/v) trichloroacetic acid with 20% (w/v) NaCl was added to stop the reaction, and the mixture was then filtered with Advantec No. 2 filter paper. Two-hundred l of the filtrate and 3.0 ml of 0.5 M sodium citrate buffer, pH 5.0, were added to the reaction mixture; 1 ml of freshly prepared ninhydrin reagent (19) was also added. The linearity of the assay was checked, and the amount of amino acid produced in the reaction mixture was determined. One katal is defined as the amount of enzyme yielding the color equivalent of 1 mol of tyrosine/s with ninhydrin reagent using protein substrates at pH 7.0 and 30°C according to previous work (19).
Protein Concentration-Protein concentration was determined using the BCA protein assay reagent (Pierce) following the manufacturer's instructions. A set of protein standards of known concentration was prepared by diluting the bovine serum albumin standard solution provided with this kit. Ten l of each standard or unknown protein sample was pipetted into the tube, and then 200 l of working reagent was added to each tube and mixed well. All tubes were incubated at 60°C for 30 min and cooled to room temperature, and the absorbance at 562 nm was measured.
N-terminal Amino Acid Sequence Analyses-Protein samples were loaded onto a RESOURCE RPC column (0.64 ϫ 3 cm, Amersham Pharmacia Biotech) and eluted with 0.1% (v/v) trifluoroacetic acid with a linear gradient of 0 -80% (v/v) acetonitrile. The peak fractions were concentrated with a TAITEC VA-500F Speed Vac and then subjected to N-terminal sequence determination on an Applied Biosystems 473A protein sequencer with a 610A data analysis system. SDS-PAGE 1 -SDS-PAGE supernatants (0.5 ml) of the E. coli transformants with wild-type and mutant dlnO genes were treated with trichloroacetic acid and centrifuged. The pellets were dissolved in 100 mM sodium phosphate buffer, pH 8.0, containing 0.01% SDS, heated, and denatured. The proteins were separated by SDS-PAGE as described by Laemmli (32) and then stained with Coomassie Brilliant Blue R-250 dissolved in 50% methanol and 9.5% acetic acid and destained in 5% methanol and 9.5% acetic acid.
CD Measurements-Samples were dialyzed in 5 mM phosphate buffer, pH 7.0, and then diluted in the same buffer to 0.2 mg of protein/ml by adjusting A 280 nm at 0.2. The CD measurements were done as described by Yamaguchi et al. (19) using a Jasco J-700 spectrophotometer. The contents of ␣-helix and ␤-structure of the enzyme were calculated by the SSE-338 program described by Yang et al. (33). The molecular mass value of 19,800 Da was used.
Zinc Blotting for the 65 Zinc Binding Ability Assay-The zinc blotting (34) for the 65 Zn binding ability assay was performed on SDS-polyacryl-amide gel for the site-directed mutant enzymes H128R, E129Q, H132R, D143N, and D164N. SDS-PAGE was performed according to the protocol described by Laemmli (32). Proteins were electrophoretically transferred to polyvinylidene difluoride membrane by the protein blotting technique, after which the filter was washed in metal-binding buffer (100 mM Tris-HCl, pH 6.8, containing 50 mM NaCl) for 3 h. The filter was probed for 1 h with 5 Ci of 65 ZnCl 2 /lane (2.71 Ci/g; 1 Ci ϭ 37 GBq; NEN Life Science Products) in 15-20 ml of metal-binding buffer. It was then washed in Saran Wrap and exposed to an imaging plate (Fuji Photo Film Co., Tokyo) for 12 h. After exposure, the imaging plate was analyzed by a BioImage BAS-2000 Analyzer System (Fuji Photo Film Co.). Proteins immobilized on the polyvinylidene difluoride membrane were detected by staining with Coomassie Brilliant Blue R-250 (0.05% in 50% methanol and 10% acetic acid); the protein staining was done in duplicate.
Atomic Absorption Spectrophotometric Analyses-Zinc analysis in the enzyme was also done by atomic absorption spectrophotometry using a Perkin-Elmer Model 3100 apparatus.
Molecular Mass Determination on Superdex 75 HR 10/30 -Molecular mass determination of the recombinant wild-type deuterolysins activated with Zn 2ϩ and trypsin was done by gel filtration with a Superdex 75 HR 10/30 column. The elution time of proteins through the column was plotted against logarithms of a molecular mass of standard proteins. Bovine serum albumin (67 kDa), ovalbumin (43 kDa), and myoglobin (17.4 kDa) were used as molecular mass standards.

RESULTS
Recombinant wild-type prodeuterolysin was expressed in E. coli and purified as described under "Experimental Procedures." The purified prodeuterolysin could be activated by 5 mM ZnCl 2 at 4°C for 24 h. A high yield of the mature deuterolysin was obtained by the activation method with ZnCl 2 ; however, the method required a lengthy incubation time. A more effective method of activating the purified proenzyme fractions was by incubation at 37°C for 30 min with an equal amount of trypsin (mol/mol), although the yield of the activation method with trypsin was lower than that of the method with ZnCl 2 . We obtained homogeneous preparations on SDS-PAGE of deuterolysin activated with ZnCl 2 and trypsin, as shown in Fig. 2. The molecular mass of the recombinant deuterolysin activated by ZnCl 2 was determined as 19,800 Da by gel filtration on Superdex 75 HR 10/30 (data not shown), which was similar to that of the native enzyme (19,000 Da) reported by Tatsumi et al. (17). The specific activity of deuterolysin activated by trypsin treatment was 0.144 katal/kg of protein with salmine as a substrate, and this was also the same specific activity as that of the native enzyme purified from A. oryzae. Activity resistance to 100°C treatment with Zn 2ϩ and inhibition with EDTA showed that the recombinant deuterolysin expressed in E. coli was similar to native deuterolysin.
The N-terminal sequence of recombinant wild-type deuterolysin activated with an equal amount (mol/mol) of trypsin was found to be Thr-Glu-Val-Thr-Asp, which is the N-terminal sequence of deuterolysin from A. oryzae (17), as shown in Table I. The result suggests that catalytic cleavage of the peptide bond Arg Ϫ1 -Thr 1 in prodeuterolysin by trypsin may occur. The Nterminal sequence of the recombinant wild-type enzyme activated with 5 mM ZnCl 2 for 24 h at 4°C, in contrast, was found to be Glu-Val-Thr-Asp, which was one amino acid residue shorter than that of native deuterolysin from A. oryzae. These results suggest that autocatalytic cleavage of the propeptide may occur ahead of threonine and that the product is trimmed by aminopeptidase(s) in bacterial culture broth. The ultraviolet CD spectrum of recombinant wild-type deuterolysin activated by trypsin predicted a conformation of 70% ␣-helix, 16% ␤-structure, and 14% random structure, which was almost identical to that of the native enzyme (71% ␣-helix and 29% ␤-structure).
The ultraviolet CD spectra of the five site-directed mutants H128R, E129Q, H132R, D143N, and D164N, were almost identical to that of wild-type deuterolysin. The contents of ␣-helix, ␤-structure, and random structure for the five mutants are shown in Table I; their N-terminal sequences were identical to that of wild-type deuterolysin activated with trypsin. The comparison of N-terminal sequences of these mutants is shown in Table I.
In the site-directed mutagenesis experiments, native, wildtype, and mutant enzymes were analyzed by SDS-PAGE following treatment with or without 5 mM Zn 2ϩ at 4°C for 24 h. With 5 mM Zn 2ϩ , native, wild-type, and active forms of the site-directed mutants E42Q, R58Q, E65Q, D80N, E86Q, D104N, H118A, D121N, and E142Q converted from the 56-kDa proenzyme to the 25-kDa mature form, whereas without Zn 2ϩ , this conversion did not occur in these nine mutants (data not shown). The results from SDS-PAGE are shown in Figs. 4 and 5. Substitutions of Tyr 106 , His 128 , Glu 129 , His 132 , Asp 143 , and Asp 164 resulted in mutant enzymes exhibiting complete loss of the converting activity from the proenzyme to a mature form of deuterolysin. The proteolytic activity for salmine hydrolysis at pH 7.0 of site-directed mutants of deuterolysin activated with 5 mM Zn 2ϩ is shown in Table II. All of the mutants of prodeuterolysin, on the other hand, converted from the proenzyme to the mature form with no or only trace activity by trypsin.
The recombinant forms of prodeuterolysin were activated with trypsin and purified to homogeneity. The purified mutant enzyme H132R showed complete loss of specific activity at pH 7.0 with salmine as a substrate. Furthermore, the purified mutant enzymes H128R and D164N also showed almost com-plete loss of activity. The relative specific activities of the mutant enzymes H128R and D164N for salmine hydrolysis were determined as 0.13 and 0.29%, respectively (Table III).
In 65 Zn binding measurements on SDS-PAGE, the recombinant site-directed mutants H128R, H132R, and D164N demonstrated a complete loss of 65 Zn-binding ability in the mutant enzymes (Fig. 6B). SDS-PAGE showed, for the two mutants H128R and H132R, protein bands with slightly faster mobilities on the gel, whereas for the E129Q, D143N, and D164N mutants, a single protein with an apparent molecular mass of 25 kDa (Fig. 6A) was observed. The faster mobility of the H128R and H132R mutants suggests that these proteins are less structurally stable than the wild-type enzyme and are susceptible to proteolytic trimming at their C termini. Mutant D164N did not incorporate 65 Zn 2ϩ into the molecule, whereas E129Q and D143N have the ability to incorporate 65 Zn 2ϩ , as do the native and wild-type enzymes (Fig. 6B and Table III). It was concluded that His 128 , His 132 , and Asp 164 provide the three Zn 2ϩ ligands in the active site of deuterolysin; it was also demonstrated that the two mutants E129Q and D143N have zinc-binding capacity. Atomic absorption spectrophotometric analysis confirmed that E129Q and D143N had 1 g atom of zinc/mol of enzyme (data not shown). Substitutions of Tyr 106 with Phe and of Asp 164 with Asn caused drastic decreases in proenzyme maturation and proteolytic activity for salmine hydrolysis at pH 7.0. However, the magnitude of the specific activity of mutant D143N was larger than that of the Zn 2ϩ ligand mutants H128R, H132R, and D164N. The relative specific activity of mutant D143N was determined as 0.90% (Table  III). Furthermore, the present site-directed mutagenesis experiments demonstrated that two carboxylic acid residues, Glu 129 and Asp 164 , and a single aromatic amino acid residue, Tyr 106 , are essential for the catalysis. Glu 129 in deuterolysin is thought to play a central role in catalytic function. We propose a new zinc-binding motif, aspzincin, defined by the HEXXH ϩ D motif in which an aspartic acid is the third ligand. DISCUSSION Earlier comparison (23) of the sequences of deuterolysin and penicillolysin with the metalloendopeptidases thermolysin and carboxypeptidase A showed that the primary structures of deuterolysin and penicillolysin have only a low degree of sequence identity to other metalloproteases. We previously assumed that His 128 , His 132 , and Glu 65 of penicillolysin corresponded to the zinc ligands in the homologous thermolysin and neutral proteinases (23). The two histidine residues in the HEXXH motif of deuterolysin from A. oryzae were confirmed to be zinc ligands of the enzyme by site-directed mutagenesis in the case of the mutants H128R and H132R. The possibility that Glu 65 is a zinc ligand in deuterolysin was ruled out, however, because mutant E65Q had showed proenzyme maturation and proteolytic activity for salmine hydrolysis at pH 7.0. The discovery of the third zinc ligand of deuterolysin was the major purpose of this paper.
It is easy to identify the crucial residue of Glu 129 in the HEXXH motif of deuterolysin. The present results from sitedirected mutagenesis confirmed that Glu 129 of deuterolysin is a catalytically crucial residue of this enzyme from A. oryzae. Furthermore, in these site-directed mutagenesis experiments, we demonstrated that two other amino acid residues, Asp 143 and Tyr 106 , were also catalytically crucial residues of the enzyme. Although the mutants E129Q and D143N disrupted the catalytic function, the 65 Zn-binding abilities of the mutants were maintained. These experiments showed that Glu 129 and Asp 143 were not the zinc ligands of the enzyme. It was therefore concluded that the three residues Glu 129 , Asp 143 , and Tyr 106 are crucial for the catalysis of deuterolysin from A. oryzae.  From the present findings, Glu 129 and Asp 143 of deuterolysin are probably in the ionized COO Ϫ form, and Tyr 106 of the enzyme is probably the binding site of substrate. Amino acid sequences of metalloendopeptidases for acyllysine bonds from G. frondosa and P. ostreatus fruiting bodies were reported by Nonaka et al. (27). They suggested that these proteases, G. frondosa and P. ostreatus metalloendopeptidases, do not have conserved third and/or fourth liganding amino acid residues seen in the metzincin or thermolysin superfamily of proteases, but belong instead to a novel zinc metalloendopeptidase superfamily. A zinc atom in thermolysin has been dem-onstrated to be bound to His 142 , His 146 , and Glu 166 by extensive studies of the tertiary structure (24). Nonaka et al. also indicated the presence of the homologous regions GTXDXXYG and AXXNXD among the sequences of six fungal metalloproteinases (deuterolysin from A. oryzae (17), MEP20 from A. flavus (25) and A. fumigatus (26), penicillolysin (23), and metalloendopeptidases from G. frondosa and P. ostreatus (27)) of the deuterolysin (EC 3.4.24.39) family, in which the potential zinc ligand residues Asp, Asn, and Tyr are conserved. They suggested that Asp, Asn, and/or Tyr in these regions is likely to be the third and/or fourth zinc ligand in these metalloproteinases and that the six fungal metalloproteinases might be classified into a novel subfamily of zinc metalloproteinases (27).
From the present results of site-directed mutagenesis and the 65 Zn-binding assay, His 128 and His 132 of deuterolysin from A. oryzae were shown to correspond to the zinc-binding sequence in thermolysin (24), whereas the third aspartate zinc ligand, Asp 164 , of deuterolysin was replaced by Glu 166 in thermolysin. Site-directed mutagenesis experiments showed that mutant D164N had no detectable catalytic activity for maturation from the proenzyme to the mature form or proteolytic activity for salmine hydrolysis (Fig. 4).
The Zn 2ϩ atom is bound to both thermolysin and carboxypeptidase A by interaction with the imidazole side chains of two His residues and with the carboxyl side chain of a Glu residue (1). A further coordination position of each Zn 2ϩ atom is occupied by a water molecule, which plays a crucial role in the catalytic activity of each enzyme (1). It is concluded that the two particular residues His 128 and His 132 are involved in binding zinc. However, the present finding showed that a unique amino acid residue, Asp 164 , is a third Zn 2ϩ ligand of deuterolysin. In deuterolysin, the spacer between His 132 and Asp 164 would be 31 instead of the 29 amino acids in thermolysin. It is interesting that the third ligand identified, Asp 164 , is only ϳ13 residues from the C terminus. This suggests that proper folding of this portion of the molecule may be critical both for zinc binding and for the catalytic activity of deuterolysin. Aspzincin, defined by the HEXXH ϩ D motif and aspartic acid as the third zinc ligand, is the proposed family name for the zinc metalloendopeptidase deuterolysin.  H128R, E129Q, H132R, D143N, and D164N on SDS-PAGE. The activation method with trypsin was used. Proteins were subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membrane. The washed filter was probed with 65 ZnCl 2 and exposed (B). Superoxide dismutase (SOD) and soybean trypsin inhibitor (STI) were used as positive and negative controls, respectively, for 65 Zn incorporation. Protein staining with Coomassie Brilliant Blue R-250 was done in duplicate (A).
ϩ ϩϩϩ a ϩ represents the proenzyme is able to convert to the mature enzyme. b ϩϩϩ, ϩϩ, and ϩ represent 70 -100, 30 -70, and 5-30% activity of the wild-type enzyme, respectively. University) for kind assistance in preparing photocopies of the manuscript.