The role of Tyr71 in Streptomyces trypsin on the
recognition mechanism of structural protein substrates
Yoshiko Uesugi*, Hirokazu Usuki, Masaki Iwabuchi and Tadashi Hatanaka
Research Institute for Biological Sciences, Okayama, Japan
Introduction
Serine proteases play key roles in physiological and
cellular functions, including protein processing, tissue
remodelling, immunity, cell differentiation and blood
clotting [1]. Serine proteases of clans SA (chymotryp-
sin-like) [2], SB (subtilisin-like) [3] and SC (a ⁄ b-hydro-
lase fold) [4] maintain a strictly conserved catalytic
site geometry comprising serine, histidine and aspartic
acid residues. They catalyse peptide bond hydrolysis,
which generally proceeds in a three-step mechanism:
the formation of an enzyme–substrate complex; acyla-
tion of the active site serine; and hydrolysis of the
acyl-enzyme intermediate [5].
Substrate recognition, especially the specificity at the
S1 site, has been studied extensively. The specificity at
this site in the chymotrypsin-like serine proteases has
been explained using the structure of the S1 pocket,
which comprises three b-sheets (residues 189–192,
214–216 and 224–228) and the oxyanion-binding site
Keywords
collagenolytic enzyme; repeat-length
independent and broad spectrum (RIBS)
in vivo DNA shuffling; serine protease;
Streptomyces; topological specificity
Correspondence
T. Hatanaka, Research Institute for
Biological Sciences, Okayama, 7549-1

ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ANS, 1-anilinonaphthalene-8-sulfonic acid; CBD, collagen-binding
domain; FITC, fluorescein isothiocyanate; LB, Luria–Bertani; MMP, mammalian matrix metalloprotease; RIBS, repeat-length independent
and broad spectrum; SGT, Streptomyces griseus trypsin; SOT, Streptomyces omiyaensis serine protease; Z-Gly-Pro-Arg-MCA,
benzyloxycarbonylglycyl-
L-prolyl-L-arginine-4-methylcoumaryl-7-amide.
5634 FEBS Journal 276 (2009) 5634–5646 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Gly193 and Ser195) in the C-terminal b-barrel
domain. Specificity is usually determined by the resi-
dues at positions 189, 216 and 226 [6,7]. For example,
the combination of Ser189, Gly216 and Gly226 creates
a deep hydrophobic pocket in the chymotrypsin
enzyme that accounts for S1 specificity. Furthermore,
Asp189, Gly216 and Gly226 create a negatively
charged S1 site that confers specificity of trypsin for
substrates containing arginine or lysine at the P1 posi-
tion [8,9]. Surface loops 1, 2 and 3 (residues 184–195,
213–228 and 169–175, respectively) are also important
for substrate specificity. The specificities of S2–Sn sites
have also been investigated using elastase [10]. How-
ever, the mechanism by which serine proteases recog-
nize the structure of protein substrates is not known.
Various structural features govern interactions between
protease and substrate, and therefore insight into the
mechanism is necessary to explain substrate recogni-
tion. Data on the topological specificities are available
only for the metalloproteinase ADAMTS (a disintegrin
and metalloproteinase with thrombospondin motifs)
and the mammalian matrix metalloproteases (MMP),
which were obtained using triple-helical and single-
stranded fluorogenic substrates [11].

pared their substrate specificities. Using type I and
type IV collagens as typical protein substrates with
different structures, we identified a key residue on the
substrate recognition site that conferred specificity for
the substrates.
Results and Discussion
Construction of chimeras using RIBS in vivo DNA
shuffling
In a previous study, we demonstrated that SOT had
wide substrate specificity for types I and IV collagens,
gelatin and casein, whereas SGT only showed high
substrate specificity towards type I collagen [12]. To
investigate which domain confers the different topolog-
ical specificities, we constructed a chimeric gene library
between SOT and SGT using RIBS in vivo DNA shuf-
fling (Fig. 1A). This system is a method of random
chimeragenesis based on the combination of highly fre-
quent deletion formation in the Escherichia coli ssb-3
strain with an rpsL-based chimera selection system.
We have demonstrated previously the substrate recog-
nition mechanism in Streptomyces phospholipase D
using this system [23–25].
We obtained various chimeras with recombination
sites widely distributed over the entire chimeric gene.
The DNA sequences of the parental sot gene and the
trypsin gene (sprT) encoding SGT (in S. griseus NBRC
13350) [21] are 82% identical. Therefore, these genes
are suitable for chimeragenesis using the RIBS in vivo
DNA shuffling system. We chose eight typical chime-
ras, as presented in Fig. 1B, for gene expression and

(corresponding to residues 52–72 of SOT) confers sub-
strate specificity. Therefore, we examined this region
further.
Identification of amino acid residue(s) related to
topological specificity
Chimera B differed from chimera C in five amino acid
residues (Fig. 3A). Therefore, we constructed four
chimera B mutants (B-1 to B-4, the primary sequence of
which is presented in Fig. 3A) and evaluated their speci-
ficities towards two collagen types. For type I collagen,
the specific activity of the chimera B mutant increased
as substituted residues accumulated (Fig. 3B). In con-
trast, the specific activity of the chimera B mutant
towards type IV collagen changed considerably between
B-3 and B-4 (Fig. 3C). These results were reflected in
collagen IV ⁄ I (Fig. 3D). Figure 3A shows that chimeras
A
P
T7
-lac
pACTI2b
(sot/Gm
r
rpsL
+
/sprT)
lacl
q
Cm
r

q
lacl
q
sot
sprT
rpsL+
SGT
223 aa
in vivo DNA shuffling
Chimeric genes
SOT
Chimera A
Chimera B
Chimera C
Chimera D
Chimera E
Chimera F
Chimera G
Chimera H
223 aa
223 aa
0 50 100 150
200
B
AB
CD
E
111213141
51 61 71 81 91
E

from Sm
r
to Sm
s
(and also Gm
s
to Gm
r
)
because the Sm
s
ribosome was reconsti-
tuted with the wild-type RpsL protein
encoded by the plasmid. The Gm
r
-rpsL
+
cassette is simultaneously deleted from the
plasmid and the cells reverse their pheno-
type from Sm
s
⁄ Gm
r
to Sm
r
⁄ Gm
s
when
recombination occurs between two homolo-
gous genes. Consequently, the intact form

substituted for Leu71 (SGT-L71P). Figure S1 shows
SDS-PAGE data from several culture supernatants of
SGT mutants. Unlike other mutants, the band at the
molecular mass of SGT-L71P was hardly observed,
but many other bands were seen. In addition, the cul-
ture supernatant had no activity. In the case of inac-
tive SOT and SGT, in which Ser172 of the catalytic
triad was substituted with alanine, many high molecu-
lar mass bands were observed (data not shown),
although these bands were not observed in active
forms. It is probable that correctly folded mutants
could hydrolyse these high molecular mass proteins.
For SGT-L71P, low molecular mass bands were also
observed at positions different from the case of active
mutants. We speculate that SGT-L71P, which was not
folded correctly, was presumably hydrolyzed by other
proteases in the culture. These results suggest that
residue 71 also affects the folding of SGT.
Five purified SGT mutants (substitution of Leu71
with tyrosine, phenylalanine, tryptophan, alanine or
histidine) showed higher activity towards type IV colla-
gen than did wild-type SGT (Fig. 4B, left). In parti-
cular, the collagen IV ⁄ I values of SGT-L71Y and
SGT-L71H were twice as high as that of SGT
(Fig. 4C, left). Furthermore, mutant SOT-Y71L
showed significantly lower specific activity towards
type IV collagen than did wild-type SOT, although
SOT-Y71L showed high activity towards type I colla-
gen, similar to SOT (Fig. 4A, B, right). From these
results, it is interesting to note that the substitution

B
40
60
80
100
120
Collagen type IV
0
20
SGT A
B C
D
E F G H SOT
Collagen IV/I
0.4
0.5
0.6
Type IV/I
C
0
0.1
0.2
0.3
Fig. 2. Comparison of the specific activities for chimeras A–H,
parental SGT and SOT towards different fluorescein-conjugated
substrates. The reactions were performed using bovine skin DQ-
collagen type I (A) and human placenta DQ-collagen type IV (B) in
50 m
M Tris ⁄ HCl (pH 8.0) containing 10 mM CaCl
2

1.4-fold higher in SGT-L71Y (90.3 lmolÆmin
)1
Æmg
)1
)
than SGT (66.1 lmolÆmin
)1
Æmg
)1
), and 2.6-fold lower
in SOT-Y71L (190.2 lmolÆmin
)1
Æmg
)1
) than SOT
(501.5 lmolÆmin
)1
Æmg
)1
). For this short peptide sub-
strate, substitution of residue 71 has little effect, unlike
the response seen with the structural protein substrates.
The k
cat
value of SGT-L71Y (51.3 s
)1
) was 1.6-fold
higher than that of SGT (33.0 s
)1
), although their K

D
E
Collagen IV/I
0.2
0.3
0.4
Type IV/I
0
0.1
B B-1 B-2 B-3 B-4 C
120
140
Collagen type I
0
20
40
60
80
100
Specific activity (U·mg
–1
)
B B-1 B-2 B-3 B-4 C
50
60
Collagen type IV
Specific activity (U·mg
–1
)
0

Conformational change of SOT and SGT induced
by the substitution of residue 71
We measured the CD and fluorescence spectra of
SGT-L71Y, SOT-Y71L, SOT and SGT to investigate
the effect of the mutation at residue 71 on the tertiary
and secondary structures of SOT and SGT. Figure 6A
shows that the CD spectra of SOT-Y71L and SGT-
L71Y were drastically different from those of wild-type
SOT and SGT. The spectra of the tryptophan fluores-
cence emissions of the wild-type and mutants were also
dramatically different (Fig. 6B, C). SOT and SGT
have five and four tryptophan residues, respectively,
that contributed to their fluorescence emission spectra.
Figure 6B, C shows that the emission maximum,
A
B
60
80
100
120
100
150
200
Collagen type I
SGT-L71X
0
20
40
SGT Y F W A G R H D N S
Specific activity (U·mg

SOT-
Y71L
SOT
SGT-L71X
SGT Y F W A G R H D N S
Collagen IV/I
Collagen IV/I
0.2
0.3
0.4
0.3
0.5
0.6
Type IV/I
0
0.1
0
0.1
0.2
SOT-
Y71L
SOT
Fig. 4. Effects of mutations on topological
specificity. Specific activities of SGT-L71X
mutants (left) and SOT-Y71L (right) towards
DQ-collagen type I (A) and DQ-collagen type
IV (B) were measured in 50 m
M Tris ⁄ HCl
(pH 8.0) containing 10 m
M CaCl

the substitution of residue 71 induced conformational
changes in the secondary and tertiary structures of
wild-type SOT and SGT without disruption of the
enzyme form. From these results, we consider that the
conformational changes induced by the substitution of
residue 71 affect the topological specificity.
New insights into substrate recognition
The reaction mechanism within the catalytic triad and
the specificities of the S1–Sn sites of chymotrypsin-like
serine protease have been studied extensively [5–9].
However, the recognition mechanism for structural
protein substrates remains unclear. Studies of cathepsin
K showed that its unique collagenolytic activity among
cathepsins is caused by a preference for arginine and
lysine at the P1 position and proline and glycine at the
P2 and P3 positions [28]; neither of these residues, espe-
cially proline, is preferred by other human cathepsins.
Previously, we have studied the residue preferences in
SOT and SGT using fluorescence energy transfer
A
Collagen type I
3000
4000
5000
0
1000
2000
SGT SGT-L71Y SOT-Y71L SOT
Specifc activity (U·mg
–1

placenta (B) in 50 m
M Tris ⁄ HCl (pH 8.0) containing 10 mM CaCl
2
,
enzyme was added and incubated at 37 °C for 30–60 min. Next, the
reaction was terminated by the addition of 1 lL of 0.2
M HCl; the
rate of increase in free amino groups was measured using the ninhy-
drin method. Clostridium histolyticum collagenase type I was also
estimated as a reference enzyme. One collagen digestion unit liber-
ates peptides from collagen by collagenase type I equivalent in nin-
hydrin colour to 1.0 l mol of leucine in 5 h at pH 7.4 at 37 °C in the
presence of CaCl
2
. Data are expressed as the mean ± SD of three
independent experiments. (C) The ratio of hydrolytic activity towards
type IV collagen to that towards type I collagen.
Table 1. Kinetic parameters for the hydrolysis of a short peptide
substrate by SOT, SGT and their mutants.
K
m
(lM) k
cat
(s
)1
) k
cat
⁄ K
m
(lM

responding to residue 89 of a-chymotrypsin numbering)
contributes to the topological specificity. This finding
has not been inferred from structural information.
The crystal structure of SGT consists of 15 b-sheets
and two a-helices [22] (Fig. 7A). SGT is divisible into
two domains formed by six antiparallel b-sheets with a
similar topology. The catalytic triad lies in the cleft
between the two domains. Figure 7A shows that resi-
due 71 in the b-sheet of the N-terminal b-barrel
domain is located adjacent to Trp83, approximately at
3A
˚
(corresponding to residue 103 of a-chymotrypsin
numbering). Therefore, we speculate that the substitu-
tion of residue 71 affects the interaction between this
residue and Trp83, and subsequently causes a change
in the local environment around catalytic Asp82. This
hypothesis is supported by the dramatic change in cat-
alytic efficiency of SOT and SGT when carrying a
mutation at residue 71 (Table 1).
Compared with other serine proteases for the struc-
ture, the positional relationship between residue 71
and the catalytic triad in SOT almost resembles that in
bovine trypsin (PDB ID: 1k1n) [32] and a-chymotryp-
sin (PDB ID: 5cha) [33]. Ile89 and Phe89 in bovine
trypsin and a-chymotrypsin, respectively, correspond
to Tyr71 of SOT. Residue 89 in these enzymes is situ-
ated a short distance from Ile103, approximately 5 A
˚
(corresponding to Trp83 of SOT). Thus, we speculate

SGT
SGT-L71Y
C
300
400
500
SGT
SGT-L71Y
300 320 340 360 380
0
100
200
Wavelen
g
th (nm)
Fluorescence intensity (arbitrary units)
B
500
SOT
100
200
300
400
SOT-Y71L
300 320 340 360 380
0
Wavelength (nm)
Fluorescence intensity (arbitrary units)
Fig. 6. Conformation of SOT, SGT and their mutants. CD spectra
(A) and tryptophan fluorescence emission spectra (B, C) of SOT,

residue probably forms hydrogen bonds with main-
chain atoms to form a protein–collagen complex.
Because the molecular size and structure of trypsin-
like serine proteases and these collagenases differ, the
domain corresponding to the CBD remains elusive.
Nevertheless, residue 71 might also contribute to colla-
gen-binding. Figure 7B shows that the residue is
located in the basic surface charged region. Thus, SOT
and SGT might possess other collagen-binding regions
that act synergistically with residue 71 to promote the
binding of the acidic surface of collagens to the basic
surface charged region of the enzyme. Studies are
underway using surface plasmon resonance analysis to
determine the role of residue 71 in collagen binding.
The elucidation of the mechanism of structural protein
substrate recognition in serine proteases should help
advance therapeutic research into the prevention and
treatment of thrombotic and neurodegenerative dis-
eases caused by ‘hard-to-degrade’ proteins.
Materials and methods
Materials
A spin column (Vivapure S; Vinascience, Sartorius AG,
Aubagne, France) and DQ-collagens (Molecular Probes
Inc., Eugene, OR, USA) were used for this study. Type I
collagen from bovine Achilles’ tendon, type IV collagen
from human placenta and Clostridium histolyticum collage-
nase type I were purchased from Sigma-Aldrich Inc.
(St Louis, MO, USA). Z-Gly-Pro-Arg-MCA was obtained
from Peptide Institute Inc. (Minoh, Osaka, Japan). All other
unspecified chemicals were of the highest purity available.

37H
D82
Y71
B
S172
D166
W83
37H
D82
Y71
A
Fig. 7. The amino acid residues related to
topological specificity and the conformation
of the three-dimensional structures. (A) The
overall structure of SOT is portrayed using
the Swiss-pdb viewer based on the crystal
structure of SGT. The key residue is indi-
cated in red (Tyr71 of SOT). The residues in
the catalytic site are also shown. (B) The
surface charge is represented using the
Swiss-pdb viewer. Acidic and basic surface
charges of SOT are shown as red and blue,
respectively.
Substrate recognition mechanism of Streptomyces trypsin Y. Uesugi et al.
5642 FEBS Journal 276 (2009) 5634–5646 ª 2009 The Authors Journal compilation ª 2009 FEBS
pACTI2b(sot ⁄ Gm
r
-rpsL
+
⁄ sprT) by electroporation. The

dase, with Streptomyces lividans 1326 as a host strain. The
chimeric gene was digested using NdeI and HindIII and
ligated into the NdeI–HindIII gap of pTONA5a to obtain
the expression vector.
Construction of expression vectors of chimera B
and C mutants
To identify the amino acid residues related to topological
specificity, we constructed chimera B and C mutants using
PCR amplification. To prepare the mutants (B-2 and B-3),
the following two mutagenic sense primers were synthesized
[the XhoI site (in italic type) was substituted with a silent
mutation]: 5¢-TCCAGTC(G fi C)TC(C fi G)AGCGCC
(G fi A, Val fi Ile)TCAAG-3¢ (corresponding to nucleotides
281–303 from sprT) and 5¢-TC(G fi C)TC(C fi G)AGC
GCC(G fi A, Val fi Ile)TCAAGGTCCGCTCCACCAAG
(G fi A, Val fi Ile)TC-3¢ (corresponding to nucleotides
286–321 from sprT). The target mutation was introduced
with primer sets of 5¢-TGCCGGTACGAAGCTTCA
GAGCGTGCG-3¢ (a reverse primer, corresponding to the
HindIII site of sprT) and each of the mutagenic primers,
using KOD-Plus (version 2, Toyobo Co. Ltd.). The partial
sot gene was amplified using PCR with a combination of a
forward primer (5¢-CATATGCAGAAGAACCGACTCG
TCC-3¢, corresponding to the NdeI site of sot) and a reverse
primer [5¢-GATGGCGCT(GCT fi CGA)GGACTGGAG
GT-3¢ for silent mutation of the XhoI site (in italic type)
and corresponding to nucleotides 284–306 from sot]. The
amplified DNA fragments were cloned into pCR-Blunt
II-TOPO (Invitrogen Corp.); the resulting plasmids were
confirmed by DNA sequencing. The plasmids representing

digested using NdeI and HindIII, and ligated into the
NdeI–HindIII gap of pTONA5a to construct the expression
vector.
Construction of SGT-L71X mutants and SOT-
Y71L
We constructed SGT-L71X mutants and SOT-Y71L to
investigate the effect of distinct residues on the recognition
of the substrates. To prepare SGT-L71X mutants, the
mutagenic gene was amplified using PCR with a combina-
tion of a forward primer (5¢-CAACATATGAAGCACT
TCCTGCGTGC-3¢, corresponding to the NdeI site of sprT)
and a reverse primer [5¢-CGGT(G fi A)CCGTTGTAGC
CGGGGGCCTG(GAG fi XXX)GACCTTG-3¢ for silent
mutation of the KpnI site (in italic type), corresponding to
nucleotides 315–349 from sprT]. When XXX was GTA,
GAA, CCA, GGC, GCC, GCG, GTG, GTC, GTT, GGA
and GGG, leucine was substituted with tyrosine, phenylala-
nine, tryptophan, alanine, glycine, arginine, histidine, aspar-
tic acid, asparagine, serine and proline, respectively. The
amplified DNA fragments were then cloned, sequenced and
digested with NdeI and KpnI. The plasmid representing the
partial sprT gene with the KpnI site described above was
digested with KpnI and HindIII. The fragments were ligated
Y. Uesugi et al. Substrate recognition mechanism of Streptomyces trypsin
FEBS Journal 276 (2009) 5634–5646 ª 2009 The Authors Journal compilation ª 2009 FEBS 5643
into the NdeI–HindIII gap of pTONA5a to construct the
expression vector.
To prepare the mutant SOT-Y71L, the mutagenic gene
was amplified using PCR with a combination of a forward
primer (5¢-CATATGCAGAAGAACCGACTCGTCC-3¢,

To confirm the active site concentrations of SOT and SGT,
we performed titration using methylumbelliferyl p-guani-
dinobenzoate. The procedure followed that of Coleman
et al. [41], except for the use of 0.1 m sodium phosphate
buffer (pH 6.8) containing 1 mm HCl. The fluorescence
intensity was monitored at k
ex
= 323 nm and
k
em
= 446 nm using a CORONA grating microplate reader
SH-8000Lab. The active site concentrations were estimated
using a standard curve for the fluorescence of 4-methylum-
belliferone. The concentrations of SOT and SGT were 40.2
and 10.6 lm; their concentrations determined by the Brad-
ford assay were estimated at 56.8 and 15.1 lm, respectively.
The active forms of SOT and SGT accounted for 70.8%
and 70.4% of total protein, respectively. From these results,
we judged that the amounts of enzymes used in the study
were correctly matched.
Assay for enzyme activity
To estimate the substrate specificity of the enzymes, the
hydrolytic activities were determined using fluorogenic
bovine skin DQ-collagen type I and human placenta
DQ-collagen type IV, as described previously [12]. The
reaction velocity was estimated from the standard curve
plotted with data obtained using fluorescein isothiocyanate
(FITC). One unit of activity was defined as the amount of
the enzyme necessary to release 1 nmol of FITC per minute
under these assay conditions.

above.
CD spectroscopy
The secondary structures of the proteins were estimated by
CD spectroscopy (J-720WI; Jasco Inc.). Proteins were dis-
solved to a final concentration of 0.1 mgÆmL
)1
in 10 mm
Tris ⁄ HCl (pH 8.0) containing 10 mm CaCl
2
. Spectra were
acquired at room temperature using a cuvette (path length,
2 mm). The spectra of the proteins, an average of 10 scans,
were corrected by subtracting the spectra of the corre-
sponding background media without protein.
Fluorescence spectroscopy
Fluorescence spectra were obtained using a spectrofluorom-
eter (F-4500; Hitachi Ltd.). All measurements were carried
out at room temperature with 0.61 lm proteins in 10 mm
Tris ⁄ HCl (pH 8.0) containing 10 mm CaCl
2
using a quartz
cuvette (path length, 2 mm). The excitation wavelength was
280 nm, and the excitation and emission slits were 5 nm.
Substrate recognition mechanism of Streptomyces trypsin Y. Uesugi et al.
5644 FEBS Journal 276 (2009) 5634–5646 ª 2009 The Authors Journal compilation ª 2009 FEBS
The emission was scanned from 290 to 400 nm. The spectra
of the proteins, an average of four scans, were corrected by
subtracting the spectra of the corresponding background
media without protein.
Acknowledgements

9 Krieger M, Kay LM & Stroud RM (1974) Structure
and specific binding of trypsin: comparison of inhibited
derivatives and a model for substrate binding. J Mol
Biol 83, 209–230.
10 Bode W, Meyer E Jr & Powers JC (1989) Human
leukocyte and porcine pancreatic elastase: X-ray crystal
structures, mechanism, substrate specificity, and
mechanism-based inhibitors. Biochemistry 28, 1951–
1963.
11 Lauer-Fields JL, Minond D, Sritharan T, Kashiwagi
M, Nagase H & Fields GB (2007) Substrate conforma-
tion modulates aggrecanase (ADAMTS-4) affinity and
sequence specificity. Suggestion of a common topologi-
cal specificity for functionally diverse proteases. J Biol
Chem 282, 142–150.
12 Uesugi Y, Arima J, Usuki H, Iwabuchi M & Hatanaka
T (2008) Two bacterial collagenolytic serine proteases
have different topological specificities. Biochim Biophys
Acta 1784, 716–726.
13 Itoi Y, Horinaka M, Tsujimoto Y, Matsui H & Watan-
abe K (2006) Characteristic features in the structure
and collagen-binding ability of a thermophilic collagen-
olytic protease from the thermophile Geobacillus
collagenovorans MO-1. J Bacteriol 188, 6572–6579.
14 Bressollier P, Letourneau F, Urdaci M & Verneuil B
(1999) Purification and characterization of a keratino-
lytic serine proteinase from Streptomyces albidoflavus.
Appl Environ Microbiol 65, 2570–2576.
15 Sugimoto S, Fujii T, Morimiya T, Johdo O & Nakam-
ura T (2007) The fibrinolytic activity of a novel protease

˚
resolution.
J Mol Biol 200, 523–551.
23 Mori K, Mukaihara T, Uesugi Y, Iwabuchi M & Hata-
naka T (2005) Repeat-length-independent broad-spectrum
shuffling, a novel method of generating a random chimera
library in vivo. Appl Environ Microbiol 71, 754–760.
24 Uesugi Y, Mori K, Arima J, Iwabuchi M & Hatanaka
T (2005) Recognition of phospholipids in Streptomyces
phospholipase D. J Biol Chem 280, 26143–26151.
25 Uesugi Y, Arima J, Iwabuchi M & Hatanaka T (2007)
C-terminal loop of Streptomyces phospholipase D has
multiple functional roles. Protein Sci 16, 197–207.
26 Lecaille F, Chowdhury S, Purisima E, Bro
¨
mme D &
Lalmanach G (2007) The S2 subsites of cathepsins K
and L and their contribution to collagen degradation.
Protein Sci 16, 662–670.
Y. Uesugi et al. Substrate recognition mechanism of Streptomyces trypsin
FEBS Journal 276 (2009) 5634–5646 ª 2009 The Authors Journal compilation ª 2009 FEBS 5645
27 Lecaille F, Choe Y, Brandt W, Li Z, Craik CS &
Bro
¨
mme D (2002) Selective inhibition of the collagen-
olytic activity of human cathepsin K by altering its
S2 subsite specificity. Biochemistry 41, 8447–
8454.
28 Choe Y, Leonetti F, Greenbaum DC, Lecaille F, Bogyo
M, Bromme D, Ellman JA & Craik CS (2006) Substrate

´
AR
(2008) Structural basis of specific tRNA aminoacylation
by a small in vitro selected ribozyme. Nature 454,
358–361.
35 Whitford PC, Gosavi S & Onuchic JN (2008) Confor-
mational transitions in adenylate kinase. Allosteric
communication reduces misligation. J Biol Chem 283,
2042–2048.
36 Di Cera E (2008) Thrombin. Mol Aspects Med 29, 203–
254.
37 Xu X, Chen Z, Wang Y, Bonewald L & Steffensen B
(2007) Inhibition of MMP-2 gelatinolysis by targeting
exodomain–substrate interactions. Biochem J 406 , 147–
155.
38 Wilson JJ, Matsushita O, Okabe A & Sakon J (2003) A
bacterial collagen-binding domain with novel calcium-
binding motif controls domain orientation. EMBO J
22, 1743–1752.
39 Hatanaka T, Onaka H, Arima J, Uraji M, Uesugi Y,
Usuki H, Nishimoto Y & Iwabuchi M (2008) pTONA5:
a hyperexpression vector in streptomycetes. Protein
Expr Purif 62, 244–248.
40 Laemmli UK (1970) Cleavage of structural proteins
during assembly of the head of bacteriophage T4.
Nature 227, 680–685.
41 Coleman PL, Latham HG & Shaw EN (1976) Some
sensitive methods for the assay of trypsin like enzymes.
Methods Enzymol 45, 12–26.
Supporting information