Two types of novel dipeptidyl aminopeptidases from Pseudomonas sp. strain WO24.

Abstract

Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyze Gly-Phe-p-nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and characterized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was estimated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE, indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000 Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicating that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of bulky, hydrophobic amino acids at P1 and P1' positions. In addition, DAP BII showed no endopeptidase activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopeptidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics, namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors, suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the previously reported DAP classes.

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JOURNAL OF BACTERIOLOGY, Nov. 1996, p. 6288–6295
0021-9193/96/$04.00?0
Copyright ? 1996, American Society for Microbiology
Vol. 178, No. 21
Two Types of Novel Dipeptidyl Aminopeptidases from
Pseudomonas sp. Strain WO24
WATARU OGASAWARA, GO KOBAYASHI, HIROFUMI OKADA,
AND YASUSHI MORIKAWA*
Department of Bioengineering, Nagaoka University of Technology,
1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan
Received 28 May 1996/Accepted 27 August 1996
Two kinds of dipeptidyl aminopeptidase I (DAP I [cathepsin C])-like activities which hydrolyze Gly-Phe-p-
nitroanilide (Gly-Phe-pNA) were detected in Pseudomonas sp. strain WO24. They were purified and charac-
terized. The isolated enzymes, named DAP BII and DAP BIII, were revealed to be homogeneous by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. DAP BII was esti-
mated to have a molecular mass of 150,000 Da by gel filtration and a subunit size of 73,000 Da by SDS-PAGE,
indicating it to be a homodimer. The molecular mass of DAP BIII was evaluated to be approximately 60,000
Da by gel filtration and 69,000 Da by SDS-PAGE, indicating that it is monomeric. The isoelectric points of DAP
BII and DAP BIII were 6.1 and 5.0, and their optimal pHs were 8.0 and 8.5 to 9.0, respectively. The result of
peptide mapping for DAP BII and DAP BIII showed that these enzymes consist of different components. Both
enzymes were completely inhibited by diisopropylphosphofluoride but not by general thiol inhibitors, indicat-
ing that they are serine proteases. DAP BII and DAP BIII hydrolyzed Gly-Phe-pNA but not Gly-Arg-pNA, both
of which are model substrates for mammalian DAP I. Despite these shared activities toward DAP I, DAP BII
released dipeptides from Ala-Ala-pNA and Lys-Ala-4-methylcoumarinamide (a substrate for DAP II), whereas
DAP BIII did not hydrolyze either of these compounds and was presumed to prefer substrates composed of
bulky, hydrophobic amino acids at P1 and P1? positions. In addition, DAP BII showed no endopeptidase
activity, whereas DAP BIII possessed the activity on N-terminally blocked peptide derivatives besides exopep-
tidase activity. Assays performed with bioactive peptides such as angiotensin I and neuromedin N as substrates
indicate that DAP BII has a considerably broader substrate specificity than DAP BIII and is able to hydrolyze
an X-Pro bond, an imido bond that few peptidases and no known DAPs can cleave. These characteristics,
namely, substrate specificities, molecular mass, pI, peptide mapping, pH optimum, and effect of inhibitors,
suggested that the two DAPs purified in this work are distinct enzymes and do not belong to any of the
previously reported DAP classes.
DAPs (dipeptidylpeptide hydrolases EC 3.4.14) catalyze the
removal of dipeptides from peptides, proteins, and artificial
substrates such as a ?NA derivative. They have been classified
into four groups according to their selectivity for particular
artificial substrates, pH optima, and catalytic classification in
mammalian tissues (15). DAP I, which is also known as ca-
thepsin C, is a relatively nonspecific cysteine protease and
exhibits significant activity on dipeptide sequences of the form
Gly-Arg- and Gly-Phe- at pH 6 (17). DAP II cleaves Lys-Ala-
?NA at a pH optimum of pH 5, and DAP III degrades Arg-
Arg-?NA at a pH optimum of 8 to 9. DAP IV cleaves Gly-
Pro-?NA at a pH optimum of 8. These enzymes in mammalian
tissues may be responsible for intracellular protein degrada-
tion, processing precursor polypeptides to active forms and
playing roles in signal transduction and cell adherence pro-
cesses. Further, K. S. Hui reported a new type of DAP from rat
brain membranes, which released basic aminoacyl dipeptides
from the N terminus of oligopeptides, such as Arg-0-Met-5-
enkephalin (8).
DAP IV in many microorganisms has been well studied (2,
9, 19, 23), but little is known concerning other types of DAP.
DAP I and II have not been found in microorganisms, except
for reports that Bacteroides ruminicola (16) and Dictyostelium
discoideum (4) may possess DAP-I like activities. In these
reports, the enzymes were not purified to homogeneity, and
their characteristics were not clarified. DAP III-like enzymes
were discovered in Saccharomyces cerevisiae (22) and D. dis-
coideum (3, 4, 7). Although these enzymes have been shown to
resemble mammalian DAP III, some differences were noted
and their molecular nature remains obscure. Recently, Murao
et al. reported a DAP from Streptomyces sp. strain WM-23 (18)
and suggested that the enzyme might be classified as mamma-
lian DAP II, because it had a hydrolytic activity on Ala-Phe-
pNA and was inhibited by PMSF.
On the other hand, Atkinson et al. discovered and purified a
novel DAP (dDAP or DAP V) from the cell-free broth of D.
discoideum Ax3 (1). They concluded that the dDAP did not
belong to any of the previously reported mammalian DAP
classes (DAP I to IV). We previously reported the identifica-
tion, purification, and characterization of a novel DAP that we
named DAP BI (DAP from bacteria, type I) from Pseudomo-
nas sp. strain WO24 (20). Purified DAP BI hydrolyzed Gly-
Arg-pNA but not Gly-Phe-pNA, both of which are model sub-
strates for DAP I, and possesses a strong activity to hydrolyze
Arg-Arg-MNA, a general substrate for DAP III. Although
DAP BI has DAP I- and III-like activities partly, it is distinct
from DAP I and III, on the basis of substrate specificity and the
effects on protease inhibitors. These results suggest that the
classification of microorganism-derived DAP is distinct from
that of mammalian DAP. From this point of view, identifica-
tion and characterization of additional novel DAPs in micro-
* Corresponding author. Phone: 81 (258) 466000. Fax: 81 (258)
468163.
6288

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organisms will serve to elucidate the relationship between the
mammalian DAP group and the microorganism DAP group.
DAPs having a broad substrate specificity such as DAP I
may serve as tools in some fields, such as the food industry (6)
and analytical biochemistry (11, 12). Furthermore, if various
DAPs could be easily isolated in quantity from microorgan-
isms, it would be feasible to examine the contribution of the
molecular structure to the mechanism of dipeptide liberation,
compared with those for aminopeptidases and endopeptidase.
In this study, another two types of DAP which hydrolyze
Gly-Phe-pNA but neither Gly-Arg-pNA nor Arg-Arg-MNA
were identified in Pseudomonas sp. strain WO24. These en-
zymes, named DAP BII and DAP BIII, respectively, were
purified and characterized. We determined that DAP BII and
BIII differed from each other and also from previously re-
ported DAPs in mammalia and microorganisms, suggesting
that they are new types of DAPs.
MATERIALS AND METHODS
Abbreviations. DAP, dipeptidyl aminopeptidase; pNA, p-nitroaniline; ?NA,
?-naphthylamine; MNA, 4-methoxy-?-naphthylamide; MCA, 4-methylcoumaryl-
7-amine; Suc, succinyl; (Phe)5, Phe-Phe-Phe-Phe-Phe; (Tyr)6, Tyr-Tyr-Tyr-Tyr-
Tyr-Tyr; MES, morpholineethanesulfonic acid; MOPS, morpholinepropanesulfonic
acid; CHES, cyclohexylaminoethanesulfonic acid; CAPS, cyclohexylaminopropane-
sulfonic acid; DFP, diisopropylphosphofluoride; PMSF, phenylmethanesulfonyl
fluoride; TLCK, N-tosyl-L-lysyl chloromethyl ketone; TPCK, N-tosyl-L-phenyl-
alanyl chloromethyl ketone; NEM, N-ethylmaleimide; E-64, L-trans-epoxysucci-
nyl-leucylamido(4-guanido)butane; HPLC, high-performance liquid chromatog-
raphy.
Materials. Arg-pNA, Phe-pNA, Gly-Arg-pNA, Gly-Phe-pNA, Ala-Ala-pNA,
Gly-Arg-MNA, Gly-Phe-?NA, Ser-Tyr-?NA, Arg-Arg-MNA, (Phe)5, (Tyr)6, ox-
idized insulin B chain, neuromedin N, mast cell degranulating peptide HR2, and
angiotensin I were obtained from Sigma Chemical Co. Ala-pNA, Pro-pNA,
Lys-Ala-MCA, Gly-Pro-pNA, Suc-Ala-Ala-pNA, and E-64 were purchased from
Peptide Institute Inc. (Osaka, Japan). PMSF, TLCK, TPCK, iodoacetate, chy-
mostatin, leupeptin, and pepstatin A were obtained from Nacalai Tesque, Inc.
(Osaka, Japan), DEAE-cellulofine A-500 was obtained from Seikagaku Co.
(Tokyo, Japan), and butyl-Sepharose 4B, phenyl-Sepharose HP, Sephacryl
S-300HR, and Q-Sepharose FF were obtained from Pharmacia Fine Chemicals
(Uppsala, Sweden). All other chemicals used were of analytical grade.
Preparation of cell extracts. Pseudomonas sp. strain WO24 was aerobically
grown in bouillon medium (0.7% meat extract, 1.0% peptone, 0.3% NaCl; pH
7.0) at 37?C (20). When the A600reached 1.3, the cells were harvested by
centrifugation at 9,500 ? g for 20 min at 4?C. Cells (130 g [wet weight]) were
obtained from 16 liters of culture. The cell extracts of Pseudomonas sp. strain
WO24 were prepared by the previously reported procedure (20).
Purification of DAP BII and DAP BIII. Purification of the enzymes was carried
out at 4?C. Enzymes having DAP I-like activity were fractionated from the cell
extracts by salting out with ammonium sulfate of 40 to 70% saturation. The
pellets were dissolved in 50 mM Tris-HCl buffer (pH 9.0) with a volume equal to
twice that of the pellets. Subsequently, the dissolved pellets were dialyzed over-
night against the same buffer (pH 9.0).
The dialyzed sample was chromatographed on a DEAE-cellulofine column
(2.5 by 20 cm) equilibrated with 50 mM Tris-HCl buffer (pH 9.0). The column
was washed with 5 bed volumes of the same buffer before gradient elution. The
fraction of the bound protein was eluted with a 1,000-ml linear gradient of 0 to
0.2 M NaCl in the equilibration buffer at a flow rate of 60 ml/h. Fractions
containing DAP I-like activity which hydrolyzed Gly-Phe-pNA were pooled.
The pooled fractions were allowed to give precipitation by the addition of solid
ammonium sulfate to 30% saturation. The enzyme solution was then applied to
a butyl-Sepharose 4B column (2.5 by 12 cm) equilibrated with 10 mM Tris-HCl
buffer (pH 9.0) containing saturated 30% ammonium sulfate. After the column
was washed with 5 bed volumes of equilibration buffer, the enzyme activity was
eluted with a linearly decreasing gradient of 30 to 0% saturation with ammonium
sulfate in the same buffer. Two peaks having DAP I-like activities were resolved
by this chromatography. Fractions containing each of the peaks were pooled
separately and concentrated with a Diaflo PM-10 ultrafiltration membrane.
The enzyme responsible for the first eluted peak of activity was named DAP
BII and is less hydrophobic than the enzyme responsible for the second eluted
peak of activity, which was named DAP BIII. The DAP BII fraction was applied
to a phenyl-SepharoseHP column (1.0 by 25 cm) which was equilibrated with 10
mM Tris-HCl buffer (pH 9.0) containing 30% saturated ammonium sulfate.
After the column was washed with the same buffer, elution was performed in a
linear concentration gradient of 30 to 0% saturation with ammonium sulfate.
The active fractions were pooled and then concentrated with a Diaflo PM-10
ultrafiltration membrane. The concentrate was applied to a Sephacryl S-300HR
column (2.5 by 100 cm) previously equilibrated with 50 mM Tris-HCl buffer (pH
9.0) containing 150 mM NaCl. The fractions containing DAP BII were collected
and dialyzed against 20 mM Tris-HCl buffer (pH 9.0) overnight. This preparation
was used for the subsequent characterization of DAP BII reported in this paper.
The concentrated active fraction of DAP BIII obtained in the manner de-
scribed above for DAP BII was applied to a Sepharose S-300HR column (2.5 by
100 cm) previously equilibrated with 50 mM Tris-HCl buffer (pH 9.0) containing
150 mM NaCl. The active fractions were pooled, dialyzed against 20 mM Tris-
HCl buffer (pH 9.0), and concentrated by ultrafiltration. The following charac-
terization of DAP BIII was used for this preparation.
Enzyme assay. We chose Gly-Phe-pNA rather than Gly-Phe-?NA as the
substrate in the routine assay because ?-naphthylamine is carcinogenic and not
suitable for routine use for assaying the enzyme (10). DAP BII and DAP BIII
activities were determined from the amounts of released pNA from Gly-Phe-
pNA according to a method reported previously (20). The incubation mixture
consisted of 100 ?l of 3 mM substrate, 500 ?l of 0.1 M Tris-HCl buffer (pH 8.0
for DAP BII and pH 9.0 for DAP BIII), 300 ?l of water, and 100 ?l of an
appropriately diluted enzyme solution, and the reaction was initiated by the
addition of the enzyme solution. After incubation at 37?C for 10 to 60 min, over
which time the reaction rate was linear, the reaction was stopped by the addition
of 50 ?l of 100% (wt/vol) trichloroacetic acid, and the extent of hydrolysis at A385
was measured. One unit of DAP activity was defined as the amount of enzyme
that liberates 1 ?mol of pNA per min at 37?C. Hydrolysis of pNA derivatives of
amino acids and peptides was carried out by the standard enzyme assay proce-
dure described above.
Enzymatic activity was also analyzed with derivatives of ?NA, 4-methoxy-
?NA, and MCAs as substrates (24) according to previously reported methods
(20), except for the assay pH (pH 8.0 for DAP BII and pH 9.0 for DAP BIII).
One unit of DAP activity was defined as the amount of enzyme that liberates 1
?mol of ?NA, MNA, or MCA per min at 37?C.
FIG. 1. DEAE-cellulofine column chromatography of the DAPs from
Pseudomonas sp. strain WO24. The chromatography was conducted as described
in Materials and Methods. Elution was performed with a linear gradient of 0 to
0.2 M NaCl (——) in 50 mM Tris-HCl buffer (pH 9.0). Enzyme activity was
assayed with Gly-Arg-pNA (F) and Gly-Phe-pNA (E) as described. Protein
concentrations were monitored by measuring A280.
FIG. 2. Butyl-Sepharose 4B column chromatography of the fractions having
Gly-Phe-pNA-hydrolyzing activity. Elution was performed with a linear gradient
of 30 to 0% with saturated ammonium sulfate (——) in 10 mM Tris-HCl (pH
9.0). Enzyme activity was assayed with Gly-Phe-pNA (E) as described. Protein
concentrations (– – –) were monitored by measuring A280.
VOL. 178, 1996 NOVEL DIPEPTIDYL AMINOPEPTIDASES FROM PSEUDOMONAS SP.6289

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All of the assays were done in triplicate, and the mean variation between
samples was approximately 5%.
The steady-state kinetic constants for Gly-Phe-pNA, Ala-Ala-pNA, and Gly-
Phe-?NA were determined under the same conditions as those described above,
except for substrate concentrations. Activities were measured by using the incu-
bation mixtures with substrate concentrations ranging from 0.01 to 3 mM. The
Kmand Vmaxvalues were obtained from Lineweaver-Burk plots and were ex-
pressed as the means of three separate experiments.
HPLC analysis. The breakdown products in the reaction mixture were sepa-
rated by reverse-phase HPLC with a Shodex octyldecyl silane C18-5B column
(0.46 by 25 cm). For Gly-Phe-pNA, the reaction was stopped after 0 to 60 min of
incubation by the addition of 250 ?l of 6% perchloric acid, and the sample was
neutralized with 75 ?l of 1 N KOH. The column was equilibrated with 18%
acetonitrile, 0.1 M sodium perchlorate, and 0.017 M boric acid, and the sample
applied was eluted with a linear gradient of acetonitrile from 18 to 42% for 30
min. For oxidized insulin B chain, the reaction mixture, which contained 100 ?g
of oxidized insulin B chain and 0.45 ?g of the purified enzyme in 200 ?l of 50 mM
Tris-HCl buffer (pH 8.0), was incubated at 37?C for 0 to 4 h. The reaction was
stopped by boiling for 5 min. The column was equilibrated with 0.1% trifluoro-
acetic acid, 10 ?l of the sample was injected, and then peptides were eluted with
a linear gradient of acetonitrile from 0 to 40% containing 0.1% trifluoroacetic
acid for 40 min. For angiotensin I, neuromedin N, and mast cell degranulating
peptide HR2, the reaction mixture was prepared and analyzed in a manner
similar to that for oxidized insulin B chain, except that 4.5 ?g of the enzyme was
used and the linear gradient of acetonitrile was 0 to 60%. HPLC was performed
at 30?C at a flow rate of 1 ml/min. The A200was monitored with a model SPD-6A
UV detector (Shimadzu Co., Kyoto, Japan).
Temperature and pH dependence of DAP BII and DAP BIII activity. The
effect of temperature on DAP activity in the range of 4 to 70?C was measured.
The enzyme mixture was equilibrated for 5 min at the temperature tested before
the addition of the enzyme solution. The optimum pH for hydrolyzing Gly-Phe-
pNA was determined in the pH range of 5.5 to 11 by using five kinds of buffer:
50 mM (each) MES (pH 5.5 to 7.0), MOPS (pH 7.0 to 8.0), tricine (pH 8.0 to
9.0), CHES (pH 9.0 to 10.0), and CAPS (pH 10.0 to 11.0). The thermostability
of the enzyme was determined under the routine assay conditions after the
enzyme solution was pretreated in 50 mM Tris-HCl buffer (pH 8.0 for DAP BII
and pH 9.0 for DAP BIII) for 30 min at the temperature tested. For measure-
ments the pH stability, the activity was determined similarly to the routine assay
except at a final concentration of 250 mM Tris-HCl buffer (pH 8.0 for DAP BII
and pH 9.0 for DAP BIII) after the enzyme solution had been preincubated for
30 min at a concentration of the above buffers of 50 mM (final concentration in
the assay mixture, 5 mM).
Inhibition of DAP BII and DAP BIII activity. The following protease inhibitors
(with final concentrations in parentheses) were added to the enzyme solution:
DFP (0.1 mM), PMSF (1 mM), TPCK (0.1 mM), TLCK (0.1 mM), chymostatin
(0.1 mM), leupeptin (0.1 mM), NEM (10 mM), iodoacetate (1 mM), E-64 (0.01
mM), pepstatin A (0.01 mM), ZnCl2(0.5 mM), CoCl2(0.5 mM), CaCl2(0.5
mM), EDTA (10 mM), and o-phenanthlorine (10 mM). After incubation at room
temperature for 30 min, remaining DAP activity was measured by routine as-
saying with Gly-Phe-pNA as the substrate.
Determination of molecular mass. The proteins were electrophoresed on
SDS-polyacrylamide slab gels according to the method described by Laemmli
with 10% polyacrylamide (14), with the following standard markers: carbonic
anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (66 kDa), and
ovotransferrin (77 kDa). The molecular mass of the native enzyme was deter-
mined on a column of Sephacryl S-300HR (2.5 by 100 cm) with the following
standard markers: thyroglobulin (670 kDa), catalase (232 kDa), gamma globulin
(158 kDa), bovine serum albumin (66 kDa), ovalbumin (44 kDa), and myoglobin
(17 kDa).
Peptide mapping on SDS-PAGE. An 8-?g amount (40 ?l) of purified DAP BII
and DAP BIII was separately mixed with 10 ?l of sample buffer containing 0.625
M Tris-HCl (pH 6.8), 50% glycerol, and 0.005% bromophenol blue, and the
resulting mixtures were boiled for 2 min. The boiled samples were incubated with
2 ?g of Staphylococcus aureus V8 protease at 37?C for 60 min. The reaction with
S. aureus V8 protease was stopped by the addition of 25 ?l of 40% 2-mercap-
toethanol and 25 ?l of 8% SDS, and the resulting reaction mixtures were heated
at 100?C for 2 min. Twenty microliters of the samples was subjected to SDS-
PAGE in a 15% polyacrylamide slab gel and subsequently silver stained.
Isoelectric focusing. The isoelectric point of DAPs was determined by isoelec-
tric focusing with 2% amphorite (pH 3.5 to 10) by using a model 3230 apparatus
according to isoelectric focusing manual of Atto Co. (Tokyo, Japan).
Protein determination. The protein concentrations in the purification steps
were determined spectrophotometrically by measuring the A280. The quantitative
estimation of protein was carried out by the Bio-Rad protein assay according to
the supplier’s manual with immunoglobulin as the standard.
RESULTS
Purification of DAP BII and DAP BIII. Figure 1 shows that
the DEAE-cellulofine column was effective for the separation
of Gly-Phe-pNA hydrolyzing activity of DAP BII and DAP
BIII from previously reported DAP BI activity (20). The butyl-
Sepharose chromatography resulted in two peaks of activity
hydrolyzing Gly-Phe-pNA (Fig. 2). Thus, the activities of hy-
drolyzing Gly-Phe-pNA were distinguished as DAP BII (first
eluted peak) and DAP BIII (second eluted peak), respectively.
We further chromatographed through a few steps for DAP BII
and DAP BIII. The purification procedures for DAP BII and
DAP BIII from Pseudomonas sp. strain WO24 are summarized
in Table 1. The overall purification of DAP BII was about
70-fold from the cell extracts with a 2.1% yield. The DAP BIII
was purified approximately 130-fold with a recovery of 2.4%.
Each of the two purified enzymes showed a single band on
SDS-PAGE (Fig. 3) and isoelectric focusing (data not shown).
It was demonstrated by HPLC that each preparation hydro-
lyzed Gly-Phe-pNA into Gly-Phe and pNA but did not hydro-
lyze Phe-pNA (Table 2), indicating that only DAP activity was
present in each preparation.
Molecular mass and isoelectric point. The subunit molecu-
lar masses of purified DAP BII and DAP BIII determined by
SDS-PAGE were 73 and 69 kDa, respectively (Fig. 3). Estima-
tion of molecular mass by a Sephacryl S-300 HR column gave
values of 150 kDa for DAP BII and 60 kDa for DAP BIII (data
not shown). These results suggest that DAP BII is a ho-
modimer and that DAP BIII is a monomer. The pIs of DAP
BII and DAP BIII were estimated to be 6.1 and 5.0, respec-
tively, by isoelectric focusing (data not shown). Although the
enzymes have similar activities of hydrolyzing Gly-Phe-pNA,
FIG. 3. SDS-PAGE of purified DAP BII and DAP BIII. Electrophoresis was
performed on an SDS-polyacrylamide gel (10% polyacrylamide) with molecular
mass protein standards (see Materials and Methods). Lanes: a, marker (Mr); b,
purified DAP BII (3 ?g of protein); c, purified DAP BIII (3 ?g of protein).
TABLE 1. Purification of DAP BII and DAP BIII from
Pseudomonas sp. strain WO24
Purification step
and DAP
Amt of
protein
(mg)
Total
activity
(U)a
Yield
(%)
Sp act
(U/mg)
Purifi-
cation
(fold)
Cell-free extract
Ammonium sulfate (50–70%)
DEAE-cellulofine
2,090
1,500
100
79.4
46.8
100
79
47
0.048
0.053
2.0
1.0
1.1
4124.0
DAP BII
Butyl-Sepharose 4B
Phenyl-Sepharose HP
Sephacryl S-300HR
4.00
2.25
0.63
6.80
6.60
2.09
6.8
6.6
2.1
1.7
2.9
3.3
35
61
70
DAP BIII
Butyl-Sepharose 4B
Sephacryl S-300HR
1.49
0.87
3.66
2.43
3.7
2.4
6.1
6.2
130
130
aThe enzyme activities were assayed with Gly-Phe-pNA for the substrate and
are expressed as micromoles of pNA released per minute.
6290OGASAWARA ET AL.J. BACTERIOL.

Page 4

they differ in molecular masses, the numbers of subunits, and
pI values. While these findings suggest that both DAPs are not
the same protein, a possibility remained that DAP BIII is a
hydrolyzed product of DAP BII.
Molecular difference between DAP BII and DAP BIII. The
peptide mappings of DAP BII and BIII are shown in Fig. 4,
which indicate that respective DAPs give different components
when treated with S. aureus V8 protease. It is concluded that
DAP BII and DAP BIII are not the same enzyme although
they have similar activities.
Temperature dependence and effect of pH on enzyme activ-
ity. The optimum temperatures for the Gly-Phe-pNA hydroly-
sis were found to be approximately 30?C for DAP BII and be-
tween 35 and 40?C for DAP BIII. Both enzymes were stable at
a temperature below 20?C for 30 min (data not shown). The opti-
mum pHs for the hydrolysis of Gly-Phe-pNA appeared to be
8.0 for DAP BII and between 8.5 and 9.0 for DAP BIII (Fig. 5).
Substrate specificity for synthetic amino acid derivatives.
Since mammalian DAPs are classified into four groups primar-
ily on the basis of relative cleavage rates and types of substrates
(15), we measured the activities of DAP BII and DAP BIII on
different synthetic amino acid derivatives typically used in aid
of the classification of DAP enzymes (Table 2). The pNA
derivatives of single amino acids were not hydrolyzed by either
of the enzymes, showing that DAP BII and DAP BIII are not
aminopeptidases. The DAPs showed an activity of releasing
Gly-Phe from Gly-Phe-pNA (substrate for DAP I) but did not
hydrolyze Gly-Arg-pNA, which was also hydrolyzed by DAP I
(15). On Gly-Phe-pNA, DAP BIII exhibited higher specific
activity than DAP BII. Gly-Phe-?NA was cleaved at unusually
higher rate than that for Gly-Phe-pNA only by DAP BIII. The
reason for this difference is not known but might be related to
the ?NA portion of the substrate, and DAP BIII might prefer
substrates composed of a bulky, hydrophobic amino acid at the
P1? position.
Ser-Tyr-?NA (also a substrate for DAP I) was hydrolyzed by
both enzymes, but the hydrolytic activity of DAP BIII was
higher than that of DAP BII. On the other hand, DAP BII
hydrolyzed Lys-Ala-MCA and Ala-Ala-pNA (a substrate for
mammalian DAP I, II, III, and IV), but the DAP BIII did not
hydrolyze these substrates. The cleavage of Lys-Ala-MCA by
DAP BII was a surprising and interesting result, since this
substrate is representative one for DAP II and is not cleaved by
DAP I (15). Thus, substrate specificities of both enzymes were
distinct from each other, and DAP BII had a broad substrate
specificity compared with that of DAP BIII.
Furthermore, DAP BII hydrolyzed Ala-Ala-pNA but not an
N-blocked Ala-Ala-pNA derivative, Suc-Ala-Ala-pNA, show-
ing that DAP BII possesses no endopeptidase activity. In con-
trast, enzymatic activities on N-terminally blocked peptide de-
rivatives which contain aromatic amino acid residues at the P1
position were observed for DAP BIII (Table 2). This indicates
that DAP BIII has an endopeptidase activity.
Kinetics constants. The Kmand Vmaxvalues of DAP BII for
Gly-Phe-pNA, Ala-Ala-pNA, and Gly-Phe-?NA were 1.7, 0.87,
and 7.2 mM and 9.4, 20, and 10 ?mol/min/mg, respectively.
The Kmand Vmaxvalues of DAP BIII for Gly-Phe-pNA and
Gly-Phe-?NA were 0.33 and 1.0 mM and 9.6 and 330 ?mol/
min/mg, respectively. The specific activities for these substrate
were lower than these Vmaxvalues, because these substrates
caused substrate inhibition (or product inhibition) at high con-
centrations.
Effect of various chemical reagents. The influences of sev-
eral agents on the activities of DAP BII and DAP BIII are
summarized in Table 3. ZnCl2(0.5 mM) and 10 mM o-phenan-
thlorine displayed significant influence on the activities of both
FIG. 4. Limited proteolysis of DAP BII and DAP BIII with S. aureus V8
protease on SDS-PAGE. One microgram each of purified DAP BII and DAP
BIII, was treated with 0.24 ?g of S. aureus V8 protease at 37?C for 60 min.
Sample were subjected to SDS-PAGE in a 15% polyacrylamide slab gel and were
subsequently silver stained. See Materials and Methods for further details.
Lanes: a, S. aureus V8 protease (0.24 ?g); b, treated DAP BII (1.0 ?g of purified
DAP BII and 0.24 ?g of V8 protease); and c, treated DAP BIII (1.0 mg of
purified DAP BIII and 0.24 ?g of V8 protease).
FIG. 5. pH dependence of the activities of DAP BII and DAP BIII. DAP BII
(——) and DAP BIII (– – –) were assayed for hydrolysis of Gly-Phe-pNA at
several pHs (see Materials and Methods). Activities are expressed relative to the
maximum values. E, MES; Ç, MOPS; ?, Tricine; m, CHES; ø, CAPS.
TABLE 2. Hydrolytic activities of DAP BII and DAP BIII on
various synthetic amino acid substrates
Substrate
Sp act (U/mg)
DAP BII DAP BIII
Ala-pNA
Phe-pNA
Arg-pNA
Pro-pNA
Gly-Pro-pNA
Gly-Phe-pNA
Gly-Arg-pNA
Ala-Ala-pNA
Suc-Ala-Ala-pNA
Gly-Arg-MNA
Arg-Arg-MNA
Gly-Phe-?NA
Ser-Tyr-?NA
Lys-Ala-MCA
Suc-Ala-Ala-Phe-MCA
Suc-Leu-Leu-Val-Tyr-MCA
0.0
0.0
0.0
0.0
0.0
3.3
0.0
9.5
0.0
0.0
0.0
1.2
2.1
0.26
0.0
0.0
0.0
0.0
0.0
0.0
0.0
6.2
0.0
0.0
0.0
0.0
0.0
150
41
0.0
1.2
0.19
VOL. 178, 1996NOVEL DIPEPTIDYL AMINOPEPTIDASES FROM PSEUDOMONAS SP. 6291

Page 5

enzymes. The DAP BII activity was reduced to 6.6% of that of
the control when it was incubated in 1.0 mM PMSF and was
completely inhibited by 0.1 mM DFP but not by leupeptin and
chymostatin. On the other hand, the DAP BIII activity was
strongly inhibited by 0.1 mM DFP, leupeptin, and chymostatin,
but the effect of PMSF was slight. Both of the enzymatic
activities were potently inhibited by TPCK, but not by TLCK.
Several cysteine protease inhibitors were observed not to in-
fluence the activities of DAP BII and DAP BIII. Addition of
0.5 mM CaCl2and 10 mM EDTA and NEM stimulated DAP
BIII activity.
Cleavage of bioactive peptides with DAP BII. Since DAP BII
was a DAP without an endopeptidase activity and possessed a
relatively broad substrate specificity, hydrolysis of oxidized in-
sulin B chain and angiotensin I with the enzyme was carried
out in order to elucidate its substrate specificity in more detail.
DAP BII catalyzed the sequential release of Phe-Val, Asn-Glu,
and His-Leu from the NH2terminus of oxidized insulin B
chain, but further cleavage was not detected (as shown in Fig.
6a). The presence of cysteic acid at the P2 position seems to be
the reason for no more cleavage. This enzyme also sequentially
released Asp-Arg, Val-Tyr, Ile-His, Pro-Phe, and His-Leu
from the N terminus of angiotensin I to degrade it completely
into dipeptides (Fig. 6b). From these results, DAP BII was
suggested to possess a broad specificity for liberating dipep-
tides. Surprisingly, release of Ile-His from angiotensin I im-
plied that DAP BII could also liberate dipeptides that were
linked to proline through an imido bond. To confirm this inter-
esting and unique possibility of scission of the X-Probond, DAP
BII was evaluated for its ability to cleave other peptides con-
taining X-Pro bond, such as neuromedin N (Lys-Ile-Pro-Tyr-
Ile-Leu) and mast cell degranulating peptide HR2 (Phe-Leu-
Pro-Leu-Ile-Leu-Gly-Lys-Leu-Val-Lys-Gly-Leu-Leu-NH2). As
shown in Fig. 7, neuromedin N was hydrolyzed into three
dipeptides including Pro-Tyr, whereas Phe-Leu and Pro-Leu
were formed at least from the peptide HR2 (data not shown),
indicating that DAP BII can hydrolyze an imido bonds and
X-Pro bonds, as well as amido bonds.
DISCUSSION
The data of the present work demonstrate that Pseudomonas
sp. strain WO24 contains two types of novel DAP, DAP BII
and DAP BIII, which hydrolyze Gly-Phe-pNA into Gly-Phe
and pNA, but not Gly-Arg-pNA, which is the substrate for
purified DAP BI from Pseudomonas sp. strain WO24 previ-
ously reported and also for mammalian DAP I. Purified DAP
BII has a molecular mass of 150 kDa, which may be a ho-
modimer, and DAP BIII is a monomer of 69 kDa. To deter-
mine whether DAP BII and DAP BIII are the same enzyme,
we performed peptide mapping of these enzymes. The peptide
mappings of DAP BII and DAP BIII with S. aureus V8 pro-
tease demonstrate that both enzymes consist of different com-
ponents.
As to the hydrolytic activities of DAP BII and DAP BIII on
various synthetic amino acid derivatives, DAP BII hydrolyzed
the substrates for DAP I (Gly-Phe-pNA, Gly-Phe-?NA, and
Ser-Tyr-?NA) and DAP II (Lys-Ala-MCA) and Ala-Ala-pNA
(substrate for DAP I-IV), whereas DAP BIII also possessed
the hydrolytic activity on the substrate for DAP I but failed to
degrade other substrates. These results suggested that the sub-
strate specificity of DAP BII may be broad compared with that
of DAP BIII. DAP BIII was presumed to prefer substrates
composed of bulky, hydrophobic amino acids at the P1 and P1?
positions, considering that the enzyme cleaved Gly-Phe-?NA
at a higher rate than did Gly-Phe-pNA and also (Phe)5and
(Tyr)6(data not shown). In addition, DAP BII showed only
DAP activity without an endopeptidase activity, while DAP
BIII exhibited an endopeptidase activity on N-blocked sub-
strates carrying aromatic amino acids at the P1 position, as well
as DAP activity. DAP BIII did not sequentially releases dipep-
tides from the N termini of oxidized insulin B chain and an-
giotensin I, because these peptides do not contain bulky and
hydrophobic amino acids at the P1 and P1? positions in N-
terminal amino acids and cut the peptides at internal sites
having these amino acid residues. Some exopeptidases (DAP I
from the bovine spleen [13] and the previously reported DAP
BI [20]) and endopeptidases (cathepsin B [21] and thermolysin
[5]) also show the reverse activities with each other. The DAP
activity of DAP BIII was appreciably higher than that of its
endopeptidase. Furthermore, DAP BIII hydrolyzed (Tyr)6to
(Tyr)2and (Tyr)4, which was followed by cleavage of (Tyr)4to
(Tyr)2, and (Tyr)3was only slightly detected. Therefore, DAP
BIII appears to be classified as a DAP.
Hydrolysis of peptide fragments with DAP BII demon-
strated that the enzyme has a considerably broad specificity for
releasing dipeptides from the N termini of peptides. Further-
more, as shown in the sequential liberation of dipeptides from
angiotensin I, neuromedin N, and mast cell degranulating pep-
tide HR2, DAP BII could cleave the X-Pro bond, on which
most peptidases are inactive. This is the first report of a DAP
capable of cleaving an X-Pro bond. Although DAP BII is
similar in broad substrate specificity to mammalian DAP I,
other enzymatic properties of DAP BII and BIII described
above are entirely different from those of the known mamma-
lian DAPs.
Various chemical reagents that affect the activity of purified
DAPs were examined. Incubation of DAP BII and DAP BIII
with cysteine protease inhibitors such as iodoacetate and E-64
had no effect on DAP activity except for NEM, which activated
only DAP BIII, suggesting that cysteine residues were not
essential for the action mechanism of these enzymes. These
enzymes were completely inhibited by DFP, providing strong
evidence of the presence of a serine residue at the active site.
TABLE 3. Effects of various chemical reagents on the hydrolysis
of Gly-Phe-pNA by DAP BII and DAP BIII
Reagent
Concn
(mM)
Relative activity (%)a
DAP BII DAP BIII
None
ZnCl2
CoCl2
CaCl2
EDTA
o-Phenanthlorine
DFP
PMSF
TPCK
TLCK
Chymostatin
Leupeptin
NEM
IAAb
E-64
Pepstatin A
100100
16
79
160
160
17
0.5
0.5
0.5
10
10
0.1
1
0.1
0.1
0.1
0.1
10
1
0.01
0.01
8.2
78
71
120
20
0.0
6.6
21
110
94
95
110
94
98
98
4.7
84
3.1
100
12
1.0
190
110
96
110
aPurified DAP BII and DAP BIII were preincubated with each reagent under
routine assay conditions at room temperature for 30 min, and the remaining
activities were assayed.
bIAA, iodoacetate.
6292 OGASAWARA ET AL.J. BACTERIOL.