Download A comparison of the amino acid sequence of the

Journal of General Microbiology (1993), 139, 245-249.
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245
A comparison of the amino acid sequence of the serine protease of the fish
pathogen Aeromonas salmonicida subsp. salmonicida with those of other
subtilisin-type enzymes relative to their substrate-binding sites
GEOFFREY
COLEMAN*
and PAULW. WHITBY
Department of Biochemistry, Nottingham University Medical School, Queen’s Medical Centre,
Nottingham NG7 2 U H , UK
(Received 23 July 1992;accepted 19 October 1992)
The amino acid sequence of the so-called 70 kDa (actually 64 kDa) serine protease secreted by the Gram-negative
fish pathogen Aevomunas salmonicida has been determined. It shows a high degree of homology with the complete
sequence of other bacterial serine proteases which, with molecular masses of approximately 30 kDa, are less than
half its size. This homology is particularly marked in regions adjacent to the catalytic triad Asp32, His64 and
Ser221 of subtilisin BPN’. Significant features of the A. salmonicida enzyme, a new member of the group of
cysteine-containingsubtilisin-type serine proteases, are the presence of six cysteine residues in the mature enzyme,
a 37 amino acid extension at the N-terminus and 215 amino acids at the C-terminus when compared with subtilisin
BPN. In addition to a number of smaller peptide insertions there is a non-aligned 32 amino acid sequence in a
position corresponding to its introduction between Lys213 and Tyr214 of subtilisin BPN’. This sequence is highly
hydrophilic, with Asp/Asn accounting for 10 of the 32 amino acids. Further, the possession of two Cys residues
separated by 24 amino acids provides the capacity for stabilizing the peptide as an externalized loop.
Introduction
Furunculosis is a disease of salmonid fish caused by the
Gram-negative bacterium Aeromonas salmonicida. A
characteristicof the disease is the appearance of furuncles
in the form of elongated swellings along the side of the
infected fish which contain liquefied muscle tissue. This
liquefaction is caused by a serine protease secreted by all
typical strains of A . salmonicida subsp. salmonicida (Fyfe
et al., 1986, 1987), in which it is considered to be one of
the two most important extracellular virulence factors
(Ellis, 1991).
A number of other bacterial species have been shown
to produce extracellular serine proteases, of which the
best known is subtilisin BPN’, secreted by Bacillus
amyloliquefaciens, for which a three-dimensional model
is available showing the arrangement of all the amino
acids (Wells & Estell, 1988). The substrate-binding site
consisting of a triad of amino acids has been characterized and similarities have been identified between all the
*Author for correspondence. Tel. (0602) 709362; fax (0602) 422225.
Abbreviation: VR, variable region.
bacterial serine proteases studied (Siezen et al., 1991), the
majority of which have molecular masses in the region of
30 kDa, considerably less than the estimated 70 kDa,
based on SDS-PAGEdata, of the A. salmonicida enzyme.
In view of this dissimilarity it was of interest to carry out
sequence studies in order to compare the arrangement of
amino acids, particularly in the region of the catalytic
triad, with those of the other bacterial serine proteases to
determine how the extra amino acids are accommodated
and how the substrate-binding site might be affected by
the doubling of the amino acid chain length of the
mature protein.
Methods
The predicted amino acid sequence of the serine protease of A.
salmonicida was derived from a nucleotide sequence (EMBL accession
no. X67043) (Whitby et al., 1992). Sequence alignments were made
using the CLUSTAL program.
Results and Discussion
The complete sequence of the mature A. salmonicida
serine protease is shown in Fig. 1. With a molecular mass
0001-7715 0 1993 SGM
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246
G . Coleman and P. W. Whitby
1
NESCTPLTGKEAGLDTGRSSAVRCLPGI NPLQDL
50
LNSGQNAFSPRGGMAGNDLNLWWAHRTEVLGQG
74
100
I NVAVVDDGLAIAHPDLADNVRPGSKNVVTGGSD
1 I3
PTPTDPDRCPRHSVSGII AAVDNSI GTLGVAPRV
150
OLQGFNLLDDNIQQLQKDWLYALGQRRHRRQPGL
200
QPELRMSLVDPEGANGLDQVQLDRLFEQRTQHAS
AAY I KAAGTAFSR I AAGNY VAQPHRNLPKL PFEN
250
SNIDPSNSNFWNLVVRAINADGVRSSYSSVGSNV
300
FLSAPGGEYGTDAPAMVTTDLPGCDMGYNRVDDP
330
STNRLHNNPQLDASCDYNGVMNGTSSATPNTTGA
350
MVLMAPYPDLSVRDLRDLLARNATRLDANQGPVQ
400
INYTAANGERRQVTGLEGWERNAAGLWYSPSYGFG
LVDVNKTQPCSRQPRTAATTGAVALAKGKGNGRS
450
PSAPSRYVGSSPTRSSTQVDQPLTVEAVQVMVSL
500
DHQRLPDLLIELVSPSGTRSVLLNPNNSLVGQSLDR
QQLGYVRTKGLRDMRMLSHKFYGEPAHGEWRLEVT
500
DVANAAAQVSLLDRRTNTRSTLTEGNNSQPGQLLD
597
WSRGYSVLGHDAARS*
Fig. 1. Amino acid sequence of the A. salmonicida mature serine
protease. The numbers refer to the amino acids immediately below and
the bold letters denote the amino acids corresponding to those of the
catalytic triad of subtilisin BPN‘. The 597 amino acids were identified
from the nucleotide sequence of the aspA gene (Whitby et al., 1992).
of 64173 Da it consists of 597 amino acids, compared
with the 275 in subtilisin BPN’ (Fig. 2). Thus, the 37 and
215 amino acid sequences Asnl-Ser37 and Glu382Ser597, respectively, project beyond the regions of
homology with the other serine proteases shown in Fig.
2. There are no obvious outstanding features of these
projections and they bear no homology with any other
sequences in the EMBL Database.
The alignment of amino acids Gly38 to Gly381 in the
A . salmonicida serine protease (Fig. 1) with the complete
sequence of subtilisin BPN’ is shown in Fig. 2, compared
with a number of other bacterial serine proteases of the
subtilisin type (Kwon et al., 1988). A high degree of
homology can be seen with the smaller molecular species
over the region of the catalytic triad of subtilisin BPN’,
with sequences being particularly well-conserved around
the key amino acids Asp32, His64 and Ser221.
Sequence homology was observed not only for the
catalytic triad but Ser125-Leu126-Glyl27, which forms
one wall of the active-site crevice of subtilisin BPN’, is
closely similar to the corresponding sequence Ser-LeuVal in the A . salmonicida enzyme, whilst Ala152-Ala153Gly154, which forms the opposite wall of the surface
crevice of subtilisin BPN’, is faithfully conserved
(Robertus et al., 1972). All the sequences shown in Fig. 2,
excluding the A . salmonicida enzyme, contain Am155 at
the end of this latter conserved segment, which helps
to stabilize the oxyanion generated in the tetrahedral
transition state (Carter & Wells, 1990). However, in the
64 kDa protease the same conserved sequence is duplicated and Asn does, in fact, appear at the end of the
duplicate sequence closer to the primary binding
site, Ala 160-Ala161-Gly 162-Asn163. These observations
suggest a similarity of tertiary structure with other serine
proteases sufficient to allow the use of presently available
three-dimensional models in the study of substrate
specificity,i.e. the nature of the preferred amino acid side
chains adjacent to the site of hydrolytic cleavage (Siezen
et al., 1991; Fersht, 1977).
It is interesting to note that replacement of Met222
with one of the non-oxidative residues Ala, Ser or Leu
led to mutant subtilisin BPN’ with activity reduced by
47-88 % but with complete resistance to oxidation (Estell
et al., 1985). The A . salmonicida enzyme is the only one
shown in Fig. 2 in which Met222 is replaced, the
replacement being Ser.
The most significant features of the A . salmonicida
enzyme revealed from optimal alignment of conserved
sequences are non-aligned 4, 6, 14 and 32 amino acid
peptide sequences between Gln185-Arg186, Va1143Ala144, Ala179-Val180 and Lys213-Tyr214 of subtilisin
BPN’, respectively. Such features are not uncommon
among the serine proteases, in which they exist as
externalized peptide loops connecting a-helices and psheets, referred to as variable regions (VRs) which may
vary in length from a single residue to 151 residues as in
Lactococcus lactis S K l l cell wall protease (Estell et al.,
1985). The structural non-equivalence of these loops may
result from amino acid additions or deletions or they
may result from loop flexibility or thermal motion (Gros
et al., 1990).
However, the novelty of the A . salmonicida protease,
as shown in Fig. 3, compared with other VR-containing
bacterial proteases of both Gram-positive and Gramnegative organisms (Siezen et al., 1991) is not that its 32
residue VR is a somewhat larger continuous sequence
than those which appear in other bacterial serine
proteases in the same position in relation to the a-helix
of the primary substrate binding site and the adjacent
extended /I-sheet structure but, more significantly, it
contains two cysteine residues separated by a 24 amino
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Serine protease amino acid sequences
AQ:
PR:
TH
BA
AM:
CA:
DY:
As
--
A T Q S P A P W B L D R I D Q R D L P L S N S Y T Y T A T B R B V N V Y V I D T 81 R T T H R E F - O -BRA
AAQTNAPWBLARI SSTSPBTSTYYYDESABQBSCVYVI D T B I EASHPEF-E- ---BRA
Y T PNOPY F S S - R Q Y B P Q K I Q - - - - - A P Q A W - D I
AEBSBAK I Al VDTBVQSNHPDLAOKVV-80 W
A - Q S - V P Y B V S Q I K - - - - - A P A L HSQOYT B S N V K V A V l D S B I D S S H P D L - - K V A -BOA
A-QS-VPYBI SQI K-----APALHSQQYTBSNVKVAVI D S B I DSSHPDL--NVR-BOA
NVV -BOA
A - Q T - V P Y B I P L I K - - - - - A D KV Q A Q 8 F K B A NV K V A V L D T 8 I Q A S HPDL
A-QT-VPYBI P L I K-----ADKVQAQOYKBANVKVOI
I DTBI AASHTDL--KVV-BOA
O Q N A F SPRBOMAONDL NLW-WA-HRT E V L B Q B I N V A V V D D B L A I A H P D L A D N V R P B S K
10
20
30
40
--
------
i
AQ:
PR:
TH:
BA
AM:
CA:
DY:
As:
AQ
PR:
TH:
BA
AM:
CA:
DY:
As:
AQ
PR:
TH:
0A
AM:
W.
DY:
As
R - V O Y D A L O O N Q Q - D - C N Q H O T H V A B T IQ------- B V T Y B V A K A V N L Y A V R V L D C N O S Q S T S Q
QMVKTYY Y S S - -R-D-ONOHQT H C A B T V O - - - - - S - B R T Y
BVAKKTQLFBVKVLDDNOSOQY ST
DF V D N D S T P- - Q - N Q NO HO T HCA 81 A A A V T N N S T 8 I - A B T A P K A S I L A V RV L D N S O SO T WTA
SMVPSETP--NFQ-D-DNSHQTHVABTVAAL-NNSI
BV-LBVAPSSALYAVKVLODAOSOQYSW
SFVPSETP--NYQ-D-OSSHOTHVABT
IA A L - N N S I B V - L B V A P S S A L Y A V K V L D S T Q S O Q Y S W
SFVABEAY--N-T-D-ONOHOTHVABTVAAL-DNTTBV-LBVAPSVSLYAVKVLNSSOSOSYSQ
S F V S B E SY - - N - T D- ONOHOT H V A B T V A A L - D N T T B V - L B V A P N V S LY A I K V L N S S O S O T Y S A
N V V T B O S D P T P T D P O R C P R H S V S - - e l IA A I - DNSl 6- T L B V A P R V Q L Q B F N L LOON1 Q Q L Q K O
50
60
70
80
90
100
-
-
-
- -
-- --
V I AOV DWV T R N - H R R P A - - - V A N - MSLOQOV - S T A L D N A V - - K N S I
-- AAOVVYAVAA
IIAQMDFVASDKN-NRNCPKOVVAS-LSLQOQYSSSVNSAAA---RlQ-----S SOV MV A V A A
V A N O I TY - A A D - Q - B A K - - - - V I S-LSLOOT VBNSBLQOAV--NYAW-----NKOSV V V A A A
I I NO1 E W - A I A - N - N M D - - - - V I N - tlSLOO P S B S A A L K A A V - D K A V - ASOVVVVAAA
I I NO1 E W - A I S - N - N M D - - - - - V I
N-MSLOOPSBSTALKTVV--DKAV-----SSOl V V V A A A
I VSOl EW-ATT-N-BMD-----VI
N-MSLOOASBSTAMKQAV--DNAY-----AROVVVVAAA
I VSOl EW-ATQ-N-BLD----V I N-HSLOOPSBSTALKQAV--DKAY
ASOI V V V A A A
WLYALQQRRHRRQPBLQ-----PELRMSLVDPEBANBLDQ-VQLDRLFEQRTQHASAAY I K - A A
110
120
130
140
150
-
- ---
------
----- - --
BNDN-ANAC-NYS--PARVAEALTVOA-------------T T S S DA
R A S F S N Y BSC V D L
BNNN-ADAR-NYS--PASEPSVCTVOA-------------S DRY DR
R S S F S N Y BSV L D I
BNAO-NTAP-NY---PAYYSNAIAVAS-------------T DQ N D N -KSSF ST Y BSVVDV
BNEO-STOSSSTVOYPOKYPSYIAVOA--------------V D S S N Q - - - R A S F S S V B P E L DV
BNEO-SSOSSSTVOYPAKYPSTIAVQA-------------V N S S N Q - - - - R A S F S S A B S E L DV
B N S O N S Q S T N T I Q Y P A KY D S V I AV QA
VDSNSW--- - R A S F S S V B A E L EV
B N S O - S S O S Q N T I OY P A K Y D S V I AV O A - ---V D S N K N - - - - R A S F S S V Q A E L E V
B T A F S R I A A O N Y V A Q P H R N L P K L P F E N S N l D P S N S N F WNL VV RA I WADQV R S S Y S S V Q S N V F L
160
170
180
190
-
-
-
---- - --- - -------- ----
M
PR:
TH:
BA
AM:
CA:
DY:
As:
AQ
PR:
TH:
BA:
AM:
CA:
DY:
As:
ABVAALY LEQNPSATPAS-VASAI LNO-ATT-ORLSOI --BSOSPNRLLYSLLSSOSQ
ABLAAYLMTLOKTTA-ASACR-Y I ADT-ANK-ODLSNI --PFOTVN-LAYNNYQA
A B V A O L L A S Q O R S - - - A S N I R A A I E N T - A D K I S O - T Q T Y WAKO RV N - - A Y K A V Q Y
A B A A A L I LSKHPNWT-NTQVRSSLQNT-TTKLOD-SFYY - 8 K Q L I NVQA--AAQ
A B A A A L I L S K HPT WT- N A Q V R D R L E S T - A T Y L O D - S F Y Y - 6 K O L I NV Q A - - A A Q
A B A A A L I LSKHPNLS-ASQVRNRLSST-ATY L O S - S F Y Y - B K O L I WVEA--AAQ
A B A A A L I L S K Y P T L S - A S Q V R N R L S S T - A T W L O D - S F Y Y - 6 K O L IN V E A - - A A Q
TBAMVLMAPY - P D L S - - V R D - L R D L L A R N A T R L - D A N Q B P V Q IN Y T A - - A N Q
230
240
250
260
270
-
-- --
Fig. 2. Comparison of the amino acid sequence of A . salmonicida serine protease with those of other subtilisin-typeserine proteases.
The sequence of the A . salmonicida enzyme (AS) is aligned with those of aqualysin 1 (AQ), proteinase K (PR), thermitase (TH),
subtilisin BPN’ (BA), subtilisin Amylosacchariticus (AM), subtilisinCarlsberg (CA) and subtilisin DY (DY). The numbering refers to
the sequence of subtilisin BPN’, and the active-siteresidues D32, H64 and S221 are indicatedby asterisks.Identical amino acids between
the A . salmonicida enzyme and others are in bold letters (Kwon et al., 1988).
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G . Coleman and P . W. Whitby
EF: S I YGPAGGYGDNYKI T G Q l DAREMMMTYYPTSLVSPLGKA
SE: DLMTI GGSY KLLDKY GKDAWLE ( 5 ) K Q S V L S T S S -
- --- -
ext p-sheet
AS: DL PGCDMGY NRVDDPSTNRLHNNPQLDASCDY NGVMNGTS
SM: G G A V N R E A Y N K G E L S L - - - - - - - - - - - - -
NPGYGNKSGTS
a-he\ 1x
Fig. 3. Comparison of variable regions adjacent to the primary
substrate binding site, Ser221, of a number of serine proteases relative
to subtilisin BPN'. The abbreviations are related to the following:
Bacillus amyloliquefaciens subtilisin BPN' (BA), Aeromonas salmonicida
serine protease (AS), Enterococcus faecalis cytolysin component A
(EF), Staphylococcus epidermidis epidermin leader protease (SE) and
Serratia marcescens extracellular serine protease (SM). The numerical
positions of amino acids in subtilisin BPN' are indicated (Siezen et al.,
1991).
acid sequence which provide the capacity for loop
stabilization by the formation of a disulphide bridge.
Computer-generated Kyte-Doolittle hydropathy plots
(Kyte & Doolittle, 1982) over a 52 amino acid sequence,
upstream from positions corresponding to amino acid
225 of the subtilisin BPN' sequence (see Fig. 2), of the
enzymes compared in Fig. 3 showed that the A .
salmonicida peptide sequence possessed by far the
greatest hydrophilicity. This may be attributed to the fact
that Asp/Asn account for 10 of the 32 residues of this
VR. Two of the smaller VRs have even greater
proportions of dibasic amino acids, thus, 3 of 6 are
Glu/Gln and 6 of 14 are Asp/Asn. It is also of interest,
and perhaps significant, that the peptide from which the
DNA probe was constructed (Whitby et al., 1992)
appears in this 32 residue sequence-a reflection of
accessibility?
The A . salmonicida enzyme is clearly a new member of
the group of cysteine-containing subtilisin-type serine
proteases, having six cysteine residues along its length
with only three in the region of the catalytic domain, of
which two are in the hydrophilic peptide. The sequence
shown in Fig. 2 accounts for 36 kDa of the 64 kDa
molecular mass of the mature enzyme, of which 6 kDa is
accountable to variable regions, the remainder being
distributed 4 kDa at the N- and 24 kDa at the Cterminus.
It is interesting to note that whilst the enzyme is
stabilized by disulphide bridges, with optimal activity at
50 "C and a pH of 9.5, its natural environment is in the
cold, and the producing organism does not grow at
temperatures above 25 "C. The preferred substrate of the
enzyme is casein but in an infected animal it destroys
musculature and produces furuncles (Finley, 1983).
In view of the role of this A . salmonicida enzyme as a
virulence factor in furunculosis and its application as a
vaccine constituent it is important that a structureantigenic function picture be built up with the identification of key immunoaccessibleepitopes ;in this regard
the hydrophilic 32 amino acid loop presents itself as one
obvious candidate.
P. W. Whitby wishes to thank the Science & Engineering Research
Council and the Scottish Office Agriculture & Fisheries Department for
a studentship.
References
CARTER,P. & WELLS,J. A. (1990). Functional interaction among
catalytic residues in subtilisin BPN'. Proteins Structure, Function and
Genetics 7 , 335-342.
ELLIS, A. E. (1991). An appraisal of the extracellular toxins of
Aeromonas salmonicida ssp. salmonicida. Journal of Fish Diseases 14,
265-277.
ESTELL,D. A., GRAYCAR,
T. P. & WELLS,J. A. (1985). Engineering an
enzyme by site-directed mutagenesis to be resistant to chemical
oxidation. Journal of Biological Chemistry 260, 651843521.
FERSHT,A. (1977). Enzyme Structure and Mechanism, pp. 16-18.
Reading & San Francisco: W. H. Freeman.
FINLEY,A. (1983). Studies on the extracellular protease of Aeromonas
salmonicida. PhD thesis, Nottingham University.
FYFE,
L., FINLEY,A., COLEMAN,
G. & MUNRO,A. L. S. (1986). A study
of the pathological effect of isolated Aeromonas salmonicida
extracellular protease on Atlantic salmon, Salmo salar L. Journal of
Fish Diseases 9, 403-409.
FYFE,L., COLEMAN,
G. & MUNRO,A. L. S. (1987). Identification of
major common extracellular proteins secreted by Aeromonas salmonicida strains isolated from diseased fish. Applied and Environmental
Microbiology 53, 722-726.
GROS,P., VAN GUNSTEREN,
W. F. & HOL, W. G. J. (1990). Inclusion
of thermal motion in crystallographic structures by restrained
molecular dynamics. Science 249, 1 149-1 152.
KWON, S.-T., TERADA,I., MATSUZAWA,
H. & OHTA, T. (1988).
Nucleotide sequence of the gene for aqualysin I (a thermophilic
alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme. European
Journal of Biochemistry 173, 491-497.
KYTE,J. & DOOLITTLE,
R. F. (1982). A simple method for displaying
the hydropathic character of a protein. Journal of Molecular Biology
157, 105-132.
ROBERTUS,
J. D., ALDEN,R. A., BIRKTOFT,
J. J., KRAUT,J., POWERS,
J. C. & WILCOX,P. E. (1972). An X-ray crystallographic study of the
binding peptide chloromethyl ketone inhibitors to subtilisin BPN'.
Biochemistry 11, 2439-2449.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 15:54:46
Serine protease amino acid sequences
SIEZEN,
R. J., DE Vos, W. M., LEUNISSEN,
J. A. M. & DIJKSTRA,
B. W.
(1991). Homology modelling and protein engineering strategy of
subtiliases, the family of subilisin-like serine proteinases. Protein
Engineering 4, 719-737.
WELLS,J. A. & ESTELL,
D. A. (1988). Subtilisin - an enzyme designed
to be engineered. Trends in Biochemical Sciences 13, 291-297.
249
WHITBY,P. W., LANDON,
M. & COLEMAN,
G . (1992). The cloning and
nucleotide sequence of the serine protease (aspA) of Aeromonas
salmonicida ssp. salmonicida. FEMS Microbiology Letters 99,
65-72.
MIC 139
17
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