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Journal of General Microbiology (1993), 139, 245-249. Printed in Great Britain 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 15:54:46 246 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 15:54:46 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 15:54:46 247 248 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 15:54:46