ANAN_ANACO
ID ANAN_ANACO Reviewed; 345 AA.
AC P80884; O22293;
DT 01-NOV-1997, integrated into UniProtKB/Swiss-Prot.
DT 27-APR-2001, sequence version 2.
DT 03-AUG-2022, entry version 92.
DE RecName: Full=Ananain {ECO:0000303|PubMed:31306685, ECO:0000303|PubMed:9355753};
DE EC=3.4.22.31 {ECO:0000269|PubMed:31306685};
DE Flags: Precursor;
GN Name=AN1 {ECO:0000303|PubMed:31306685, ECO:0000312|EMBL:CAA05487.1};
OS Ananas comosus (Pineapple) (Ananas ananas).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; Liliopsida; Poales; Bromeliaceae;
OC Bromelioideae; Ananas.
OX NCBI_TaxID=4615;
RN [1]
RP NUCLEOTIDE SEQUENCE [MRNA].
RC STRAIN=cv. Smooth Cayenne {ECO:0000312|EMBL:CAA05487.1};
RC TISSUE=Stem {ECO:0000312|EMBL:CAA05487.1};
RA Robertson C.E., Goodenough P.W.;
RT "Cloning and expression of ananain gene from pineapple.";
RL Submitted (NOV-1997) to the EMBL/GenBank/DDBJ databases.
RN [2]
RP PROTEIN SEQUENCE OF 123-338, AND MASS SPECTROMETRY.
RC TISSUE=Stem {ECO:0000303|PubMed:9355753};
RX PubMed=9355753; DOI=10.1042/bj3270199;
RA Lee K.L., Albee K.L., Bernasconi R.J., Edmunds T.;
RT "Complete amino acid sequence of ananain and a comparison with stem
RT bromelain and other plant cysteine proteases.";
RL Biochem. J. 327:199-202(1997).
RN [3] {ECO:0000312|PDB:6MIS, ECO:0000312|PDB:6OKJ}
RP X-RAY CRYSTALLOGRAPHY (1.73 ANGSTROMS) OF 123-337 AND IN COMPLEX WITH
RP INHIBITOR, FUNCTION, CATALYTIC ACTIVITY, ACTIVITY REGULATION,
RP BIOPHYSICOCHEMICAL PROPERTIES, SUBSTRATE SPECIFICITY, TISSUE SPECIFICITY,
RP AND DISULFIDE BONDS.
RX PubMed=31306685; DOI=10.1016/j.biochi.2019.07.011;
RA Yongqing T., Wilmann P.G., Pan J., West M.L., Brown T.J., Mynott T.,
RA Pike R.N., Wijeyewickrema L.C.;
RT "Determination of the crystal structure and substrate specificity of
RT ananain.";
RL Biochimie 166:194-202(2019).
CC -!- FUNCTION: Cysteine protease. Displays a high level of diversity in
CC substrate specificity at the P1-P1' cleavage site. A hydrophilic P1
CC residue is preferred, with Gln or Arg strongly preferred. Favors an
CC Ile/Leu residue at the P2 position of substrates, with an overall
CC higher preference for Leu. The optimal tripeptide for cleavage is Pro-
CC Leu-Gln, with cleavage occurring after the Gln residue. Another optimal
CC tripeptide is Val-Leu-Arg, which may imply that a hydrophobic residue
CC at the P3 position of substrates is preferred.
CC {ECO:0000269|PubMed:31306685}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=Hydrolysis of proteins with broad specificity for peptide
CC bonds. Best reported small molecule substrate Bz-Phe-Val-Arg-|-NHMec,
CC but broader specificity than fruit bromelain.; EC=3.4.22.31;
CC Evidence={ECO:0000269|PubMed:31306685};
CC -!- ACTIVITY REGULATION: Strongly inhibited by chicken egg-white cystatin
CC (Probable). Inhibited by iodoacetamide and the active-site-directed
CC inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64)
CC (PubMed:31306685). {ECO:0000269|PubMed:31306685, ECO:0000305}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=10.5 uM for synthetic 7-amino-4-methylcoumarin (AMC) substrate Z-
CC Phe-Val-Arg-AMC (at pH 5.0 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:31306685};
CC KM=18.0 uM for synthetic 7-amino-4-methylcoumarin (AMC) substrate Z-
CC Phe-Arg-AMC (at pH 5.0 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:31306685};
CC KM=198 uM for synthetic 7-amino-4-methylcoumarin (AMC) substrate Z-
CC Arg-Arg-AMC (at pH 5.0 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:31306685};
CC KM=5.92 uM for tripeptidyl substrate Pro-Leu-Gln (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=4.29 uM for tripeptidyl substrate Val-Leu-Arg (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=3.94 uM for tripeptidyl substrate Pro-Leu-Arg (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=4.15 uM for tripeptidyl substrate Ala-Leu-Arg (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=5.36 uM for tripeptidyl substrate Val-Leu-Lys (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=6.94 uM for tripeptidyl substrate Pro-Leu-Lys (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=5.85 uM for tripeptidyl substrate Ala-Leu-Lys (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC KM=4.50 uM for tripeptidyl substrate Pro-Leu-Asn (at pH 7.4 and 37
CC degrees Celsius) {ECO:0000269|PubMed:31306685};
CC Note=kcat is 13.9 sec(-1) for Z-Phe-Val-Arg-AMC. kcat is 1.58 sec(-1)
CC for Z-Phe-Arg-AMC. kcat is 0.16 sec(-1) for Z-Arg-Arg-AMC. kcat is
CC 9.80 sec(-1) for Pro-Leu-Gln. kcat is 7.13 sec(-1) for Val-Leu-Arg.
CC kcat is 5.62 sec(-1) for Pro-Leu-Arg. kcat is 4.96 sec(-1) for Ala-
CC Leu-Arg. kcat is 6.29 sec(-1) for Val-Leu-Lys. kcat is 6.08 sec(-1)
CC for Pro-Leu-Lys. kcat is 3.58 sec(-1) for Ala-Leu-Lys. kcat is 0.36
CC sec(-1) for Pro-Leu-Asn. {ECO:0000269|PubMed:31306685};
CC -!- TISSUE SPECIFICITY: Stem (at protein level).
CC {ECO:0000269|PubMed:31306685}.
CC -!- MASS SPECTROMETRY: Mass=23478; Method=Electrospray;
CC Evidence={ECO:0000269|PubMed:9355753};
CC -!- SIMILARITY: Belongs to the peptidase C1 family. {ECO:0000255|PROSITE-
CC ProRule:PRU10088, ECO:0000255|PROSITE-ProRule:PRU10089,
CC ECO:0000255|PROSITE-ProRule:PRU10090}.
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DR EMBL; AJ002477; CAA05487.1; -; mRNA.
DR PIR; T07839; T07839.
DR RefSeq; XP_020089326.1; XM_020233737.1.
DR PDB; 6MIS; X-ray; 1.98 A; A/B=123-337.
DR PDB; 6OKJ; X-ray; 1.73 A; A/B=123-337.
DR PDB; 6Y6L; X-ray; 1.30 A; A/B=123-338.
DR PDB; 6YCB; X-ray; 1.26 A; A/B=123-338.
DR PDB; 6YCC; X-ray; 1.30 A; A/B=123-338.
DR PDB; 6YCD; X-ray; 1.35 A; A/B=123-338.
DR PDBsum; 6MIS; -.
DR PDBsum; 6OKJ; -.
DR PDBsum; 6Y6L; -.
DR PDBsum; 6YCB; -.
DR PDBsum; 6YCC; -.
DR PDBsum; 6YCD; -.
DR AlphaFoldDB; P80884; -.
DR SMR; P80884; -.
DR Allergome; 694; Ana c 2.
DR MEROPS; C01.026; -.
DR GeneID; 109710925; -.
DR GO; GO:0008234; F:cysteine-type peptidase activity; IEA:UniProtKB-KW.
DR GO; GO:0006508; P:proteolysis; IEA:UniProtKB-KW.
DR CDD; cd02248; Peptidase_C1A; 1.
DR InterPro; IPR038765; Papain-like_cys_pep_sf.
DR InterPro; IPR025661; Pept_asp_AS.
DR InterPro; IPR000169; Pept_cys_AS.
DR InterPro; IPR025660; Pept_his_AS.
DR InterPro; IPR000668; Peptidase_C1A_C.
DR InterPro; IPR039417; Peptidase_C1A_papain-like.
DR InterPro; IPR013201; Prot_inhib_I29.
DR Pfam; PF08246; Inhibitor_I29; 1.
DR Pfam; PF00112; Peptidase_C1; 1.
DR PRINTS; PR00705; PAPAIN.
DR SMART; SM00848; Inhibitor_I29; 1.
DR SMART; SM00645; Pept_C1; 1.
DR SUPFAM; SSF54001; SSF54001; 1.
DR PROSITE; PS00640; THIOL_PROTEASE_ASN; 1.
DR PROSITE; PS00139; THIOL_PROTEASE_CYS; 1.
DR PROSITE; PS00639; THIOL_PROTEASE_HIS; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Direct protein sequencing; Disulfide bond; Hydrolase;
KW Protease; Signal; Thiol protease; Zymogen.
FT SIGNAL 1..24
FT /evidence="ECO:0000255"
FT PROPEP 25..122
FT /evidence="ECO:0000255, ECO:0000305|PubMed:9355753"
FT /id="PRO_0000026400"
FT CHAIN 123..345
FT /note="Ananain"
FT /evidence="ECO:0000305|PubMed:9355753"
FT /id="PRO_0000026401"
FT ACT_SITE 147
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU10088,
FT ECO:0000305|PubMed:31306685"
FT ACT_SITE 279
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU10089,
FT ECO:0000305|PubMed:31306685"
FT ACT_SITE 300
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU10090"
FT DISULFID 144..184
FT /evidence="ECO:0000269|PubMed:31306685,
FT ECO:0007744|PDB:6MIS, ECO:0007744|PDB:6OKJ"
FT DISULFID 178..217
FT /evidence="ECO:0000269|PubMed:31306685,
FT ECO:0007744|PDB:6MIS, ECO:0007744|PDB:6OKJ"
FT DISULFID 273..325
FT /evidence="ECO:0000269|PubMed:31306685,
FT ECO:0007744|PDB:6MIS, ECO:0007744|PDB:6OKJ"
FT CONFLICT 291
FT /note="S -> A (in Ref. 2; AA sequence)"
FT /evidence="ECO:0000305"
FT CONFLICT 324
FT /note="L -> I (in Ref. 2; AA sequence)"
FT /evidence="ECO:0000305"
FT TURN 129..133
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 143..145
FT /evidence="ECO:0007829|PDB:6YCD"
FT HELIX 147..164
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 172..178
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 179..181
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 183..185
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 189..198
FT /evidence="ECO:0007829|PDB:6YCB"
FT TURN 205..207
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 229..233
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 239..246
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 251..255
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 259..263
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 266..269
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 279..288
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 294..299
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 311..315
FT /evidence="ECO:0007829|PDB:6YCB"
FT HELIX 324..326
FT /evidence="ECO:0007829|PDB:6YCB"
FT STRAND 332..335
FT /evidence="ECO:0007829|PDB:6YCB"
SQ SEQUENCE 345 AA; 38248 MW; FAF2989080174D87 CRC64;
MTSKVQLVFL FLFLCVMWAS PSAASCDEPS DPMMKQFEEW MAEYGRVYKD NDEKMLRFQI
FKNNVNHIET FNNRNGNSYT LGINQFTDMT NNEFVAQYTG LSLPLNIKRE PVVSFDDVDI
SSVPQSIDWR DSGAVTSVKN QGRCGSCWAF ASIATVESIY KIKRGNLVSL SEQQVLDCAV
SYGCKGGWIN KAYSFIISNK GVASAAIYPY KAAKGTCKTN GVPNSAYITR YTYVQRNNER
NMMYAVSNQP IAAALDASGN FQHYKRGVFT GPCGTRLNHA IVIIGYGQDS SGKKFWIVRN
SWGAGWGEGG YIRLARDVSS SFGLCGIAMD PLYPTLQSGP SVEVI
