ASPG_PYRFU
ID ASPG_PYRFU Reviewed; 326 AA.
AC Q8TZE8;
DT 10-FEB-2021, integrated into UniProtKB/Swiss-Prot.
DT 01-JUN-2002, sequence version 1.
DT 03-AUG-2022, entry version 109.
DE RecName: Full=L-asparaginase {ECO:0000303|PubMed:20370616};
DE EC=3.5.1.1 {ECO:0000269|PubMed:20370616, ECO:0000269|PubMed:22166247};
GN OrderedLocusNames=PF2047 {ECO:0000312|EMBL:AAL82171.1};
OS Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1).
OC Archaea; Euryarchaeota; Thermococci; Thermococcales; Thermococcaceae;
OC Pyrococcus.
OX NCBI_TaxID=186497;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 43587 / DSM 3638 / JCM 8422 / Vc1;
RX PubMed=10430560; DOI=10.1093/genetics/152.4.1299;
RA Maeder D.L., Weiss R.B., Dunn D.M., Cherry J.L., Gonzalez J.M.,
RA DiRuggiero J., Robb F.T.;
RT "Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P.
RT horikoshii inferred from complete genomic sequences.";
RL Genetics 152:1299-1305(1999).
RN [2]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND SUBUNIT.
RX PubMed=20370616; DOI=10.1134/s0006297910030144;
RA Bansal S., Gnaneswari D., Mishra P., Kundu B.;
RT "Structural stability and functional analysis of L-asparaginase from
RT Pyrococcus furiosus.";
RL Biochemistry (Mosc.) 75:375-381(2010).
RN [3]
RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF THR-53;
RP TYR-273 AND LYS-274, AND BIOTECHNOLOGY.
RX PubMed=22166247; DOI=10.1096/fj.11-191254;
RA Bansal S., Srivastava A., Mukherjee G., Pandey R., Verma A.K., Mishra P.,
RA Kundu B.;
RT "Hyperthermophilic asparaginase mutants with enhanced substrate affinity
RT and antineoplastic activity: structural insights on their mechanism of
RT action.";
RL FASEB J. 26:1161-1171(2012).
RN [4]
RP DOMAIN.
RX PubMed=23551356; DOI=10.1111/febs.12271;
RA Tomar R., Garg D.K., Mishra R., Thakur A.K., Kundu B.;
RT "N-terminal domain of Pyrococcus furiosus L-asparaginase functions as a
RT non-specific, stable, molecular chaperone.";
RL FEBS J. 280:2688-2699(2013).
RN [5]
RP SUBUNIT, DOMAIN, AND BIOPHYSICOCHEMICAL PROPERTIES.
RX PubMed=25862541; DOI=10.1007/s00792-015-0748-z;
RA Garg D.K., Tomar R., Dhoke R.R., Srivastava A., Kundu B.;
RT "Domains of Pyrococcus furiosus L-asparaginase fold sequentially and
RT assemble through strong intersubunit associative forces.";
RL Extremophiles 19:681-691(2015).
RN [6] {ECO:0007744|PDB:4NJE, ECO:0007744|PDB:4Q0M, ECO:0007744|PDB:4RA6, ECO:0007744|PDB:4RA9}
RP X-RAY CRYSTALLOGRAPHY (2.05 ANGSTROMS) OF 1-182 AND 202-326 OF APOENZYME
RP AND IN COMPLEX WITH L-ASPARTATE, DOMAIN, REACTION MECHANISM, AND ACTIVE
RP SITE.
RX PubMed=25478837; DOI=10.1107/s1399004714023414;
RA Tomar R., Sharma P., Srivastava A., Bansal S., Ashish F., Kundu B.;
RT "Structural and functional insights into an archaeal L-asparaginase
RT obtained through the linker-less assembly of constituent domains.";
RL Acta Crystallogr. D 70:3187-3197(2014).
RN [7] {ECO:0007744|PDB:5CBP}
RP X-RAY CRYSTALLOGRAPHY (2.36 ANGSTROMS) OF 1-182 AND 202-326.
RA Sharma P., Yadav S.P., Tomar R., Kundu B., Ashish F.;
RT "Crystal Structure of Conjoint Pyrococcus furiosus L-asparaginase at 37
RT degree C.";
RL Submitted (JUL-2015) to the PDB data bank.
RN [8] {ECO:0007744|PDB:5B5U}
RP X-RAY CRYSTALLOGRAPHY (2.61 ANGSTROMS) OF 1-178 AND 202-326.
RA Sharma P., Tomar R., Nath S.K., Kundu B., Ashish F.;
RT "Crystal structure of truncated Pyrococcus furiosus L-asparaginase with
RT peptide.";
RL Submitted (MAY-2016) to the PDB data bank.
RN [9] {ECO:0007744|PDB:5B74}
RP X-RAY CRYSTALLOGRAPHY (2.04 ANGSTROMS) OF 1-182 AND 202-326.
RA Sharma P., Tomar R., Nath S.K., Kundu B., Ashish F.;
RT "Crystal structure of conjoined Pyrococcus furiosus L-asparaginase with
RT peptide.";
RL Submitted (JUN-2016) to the PDB data bank.
CC -!- FUNCTION: Catalyzes the hydrolysis of L-asparagine into L-aspartate and
CC ammonia. Displays no glutaminase activity, a highly desirable
CC therapeutic property. {ECO:0000269|PubMed:20370616,
CC ECO:0000269|PubMed:22166247}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=H2O + L-asparagine = L-aspartate + NH4(+);
CC Xref=Rhea:RHEA:21016, ChEBI:CHEBI:15377, ChEBI:CHEBI:28938,
CC ChEBI:CHEBI:29991, ChEBI:CHEBI:58048; EC=3.5.1.1;
CC Evidence={ECO:0000269|PubMed:20370616, ECO:0000269|PubMed:22166247};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=12 mM for L-asparagine (at pH 9.0 and 80 degrees Celsius)
CC {ECO:0000269|PubMed:20370616};
CC KM=12.1 mM for L-asparagine (at pH 8.2 and 80 degrees Celsius)
CC {ECO:0000269|PubMed:22166247};
CC KM=8.12 mM for L-asparagine (at pH 7.4 and 37 degrees Celsius)
CC {ECO:0000269|PubMed:22166247};
CC Note=kcat is 870 sec(-1) (at pH 9.0 and 80 degrees Celsius)
CC (PubMed:20370616). kcat is 888.6 sec(-1) (at pH 8.2 and 80 degrees
CC Celsius) (PubMed:22166247). kcat is 17.98 sec(-1) (at pH 7.4 and 37
CC degrees Celsius) (PubMed:22166247). {ECO:0000269|PubMed:20370616,
CC ECO:0000269|PubMed:22166247};
CC pH dependence:
CC Optimum pH is 9.0. {ECO:0000269|PubMed:20370616};
CC Temperature dependence:
CC Optimum temperature is around 90 degrees Celsius (PubMed:20370616).
CC Thermostable (PubMed:20370616, PubMed:25862541). The extreme thermal
CC stability of this enzyme is due to the presence of high intersubunit
CC associative forces supported by extensive H-bonding and ionic
CC interactions network (PubMed:25862541). {ECO:0000269|PubMed:20370616,
CC ECO:0000269|PubMed:25862541};
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:20370616,
CC ECO:0000269|PubMed:25862541}.
CC -!- DOMAIN: Consists of distinct N- and C-terminal domains (NPfA and CPfA,
CC respectively), connected by a linker. NPfA acts as specific internal
CC chaperone by mediating folding of its own C-domain (CPfA), but also
CC functions as a non-specific molecular chaperone by preventing
CC aggregation of unrelated proteins (PubMed:23551356, PubMed:25862541).
CC The linker is dispensable since domains of this enzyme can assemble
CC without the linker into a conjoined tetrameric form that exhibits
CC higher activity than the parent enzyme, while each domain is inactive
CC independently. The structural integrity of the conjoined enzyme is
CC maintained through domain-domain interactions rather than the covalent
CC linker (PubMed:25478837). {ECO:0000269|PubMed:23551356,
CC ECO:0000269|PubMed:25478837, ECO:0000269|PubMed:25862541}.
CC -!- BIOTECHNOLOGY: Absence of glutaminase activity, higher stability, and
CC mesoactivity makes P.furiosus L-asparaginase and its mutants promising
CC candidates for clinical as well as industrial usage.
CC {ECO:0000269|PubMed:22166247}.
CC -!- MISCELLANEOUS: Microbial L-asparaginase is considered as an important
CC biopharmaceutical drug enzyme in the treatment of childhood acute
CC lymphoblastic leukemia (ALL). It functions by reducing the availability
CC of circulatory L-asparagine to tumor cells. The principle behind the
CC cytotoxic effect of L-asparaginase stems from the fact that the
CC leukemic lymphoblastic tumor cells and other blood tumor cells are
CC auxotrophs towards the L-asparagine and exhibit low L-asparagine
CC synthetase (ASNS) activity for de novo synthesis of L-asparagine.
CC Therefore, these tumor cells are required for exogenous supply of L-
CC asparagine for proliferation and survival. L-Asparaginase has also been
CC used for making a diagnostic biosensor as the amount of ammonia
CC produced by the action of the enzyme directly correlates to the level
CC of L-asparagine in a patient's blood (Probable). P.furiosus L-
CC asparaginase and its mutants show significant killing of cultured human
CC leukemic cell lines HL60, MCF7, and K562 (PubMed:22166247).
CC {ECO:0000269|PubMed:22166247, ECO:0000305}.
CC -!- SIMILARITY: Belongs to the asparaginase 1 family. {ECO:0000305}.
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DR EMBL; AE009950; AAL82171.1; -; Genomic_DNA.
DR RefSeq; WP_011013191.1; NZ_CP023154.1.
DR PDB; 4NJE; X-ray; 2.50 A; A=1-182, B=202-326.
DR PDB; 4Q0M; X-ray; 2.23 A; A=1-326.
DR PDB; 4RA6; X-ray; 2.50 A; A/P=1-182, B/Q=202-326.
DR PDB; 4RA9; X-ray; 2.05 A; A=1-182, B=202-326.
DR PDB; 5B5U; X-ray; 2.61 A; A=1-178, B=202-326.
DR PDB; 5B74; X-ray; 2.04 A; A=1-182, B=202-326.
DR PDB; 5CBP; X-ray; 2.36 A; A=1-182, B=202-326.
DR PDBsum; 4NJE; -.
DR PDBsum; 4Q0M; -.
DR PDBsum; 4RA6; -.
DR PDBsum; 4RA9; -.
DR PDBsum; 5B5U; -.
DR PDBsum; 5B74; -.
DR PDBsum; 5CBP; -.
DR AlphaFoldDB; Q8TZE8; -.
DR SMR; Q8TZE8; -.
DR STRING; 186497.PF2047; -.
DR EnsemblBacteria; AAL82171; AAL82171; PF2047.
DR GeneID; 41713869; -.
DR KEGG; pfu:PF2047; -.
DR PATRIC; fig|186497.12.peg.2124; -.
DR eggNOG; arCOG01924; Archaea.
DR HOGENOM; CLU_019134_2_3_2; -.
DR OMA; AIVPKLM; -.
DR OrthoDB; 69694at2157; -.
DR PhylomeDB; Q8TZE8; -.
DR BRENDA; 3.5.1.1; 5243.
DR Proteomes; UP000001013; Chromosome.
DR GO; GO:0004067; F:asparaginase activity; IEA:UniProtKB-EC.
DR GO; GO:0006520; P:cellular amino acid metabolic process; IEA:InterPro.
DR CDD; cd08963; L-asparaginase_I; 1.
DR Gene3D; 3.40.50.1170; -; 1.
DR Gene3D; 3.40.50.40; -; 1.
DR InterPro; IPR006033; AsnA_fam.
DR InterPro; IPR036152; Asp/glu_Ase-like_sf.
DR InterPro; IPR006034; Asparaginase/glutaminase-like.
DR InterPro; IPR027475; Asparaginase/glutaminase_AS2.
DR InterPro; IPR040919; Asparaginase_C.
DR InterPro; IPR027473; L-asparaginase_C.
DR InterPro; IPR041725; L-asparaginase_I.
DR InterPro; IPR027474; L-asparaginase_N.
DR InterPro; IPR037152; L-asparaginase_N_sf.
DR Pfam; PF00710; Asparaginase; 1.
DR Pfam; PF17763; Asparaginase_C; 1.
DR PIRSF; PIRSF001220; L-ASNase_gatD; 1.
DR PRINTS; PR00139; ASNGLNASE.
DR SMART; SM00870; Asparaginase; 1.
DR SUPFAM; SSF53774; SSF53774; 1.
DR TIGRFAMs; TIGR00519; asnASE_I; 1.
DR PROSITE; PS00917; ASN_GLN_ASE_2; 1.
DR PROSITE; PS51732; ASN_GLN_ASE_3; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Hydrolase; Reference proteome.
FT CHAIN 1..326
FT /note="L-asparaginase"
FT /id="PRO_0000451748"
FT DOMAIN 1..319
FT /note="Asparaginase/glutaminase"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01068"
FT ACT_SITE 11
FT /note="Nucleophile; O-isoaspartyl threonine intermediate"
FT /evidence="ECO:0000305|PubMed:25478837"
FT ACT_SITE 83
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:25478837"
FT ACT_SITE 84
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:25478837"
FT ACT_SITE 154
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:25478837"
FT ACT_SITE 273
FT /note="Charge relay system"
FT /evidence="ECO:0000305|PubMed:25478837"
FT BINDING 11
FT /ligand="L-aspartate"
FT /ligand_id="ChEBI:CHEBI:29991"
FT /evidence="ECO:0000269|PubMed:25478837,
FT ECO:0007744|PDB:4NJE"
FT BINDING 51..52
FT /ligand="L-aspartate"
FT /ligand_id="ChEBI:CHEBI:29991"
FT /evidence="ECO:0000269|PubMed:25478837,
FT ECO:0007744|PDB:4NJE"
FT BINDING 83..84
FT /ligand="L-aspartate"
FT /ligand_id="ChEBI:CHEBI:29991"
FT /evidence="ECO:0000269|PubMed:25478837,
FT ECO:0007744|PDB:4NJE"
FT BINDING 109
FT /ligand="L-aspartate"
FT /ligand_id="ChEBI:CHEBI:29991"
FT /evidence="ECO:0007744|PDB:4NJE"
FT MUTAGEN 53
FT /note="T->Q: Shows improved enzymatic properties at
FT physiological conditions (pH 7.4 and 37 degrees Celsius) as
FT compared to the wild type with a 2-fold increase in
FT catalytic efficiency. Shows higher and significant killing
FT of human leukemic and breast carcinoma cell lines as
FT compared to the E.coli L-asparaginase."
FT /evidence="ECO:0000269|PubMed:22166247"
FT MUTAGEN 273
FT /note="Y->A: 95% reduction in activity compared to wild
FT type."
FT /evidence="ECO:0000269|PubMed:22166247"
FT MUTAGEN 274
FT /note="K->E: Shows improved enzymatic properties at
FT physiological conditions (pH 7.4 and 37 degrees Celsius) as
FT compared to the wild type with a 2.6-fold increase in
FT catalytic efficiency. Shows higher and significant killing
FT of human leukemic and breast carcinoma cell lines as
FT compared to the E.coli L-asparaginase."
FT /evidence="ECO:0000269|PubMed:22166247"
FT STRAND 3..10
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 11..13
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 14..16
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 18..20
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 22..24
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 27..34
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 41..50
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 52..54
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 57..70
FT /evidence="ECO:0007829|PDB:5B74"
FT TURN 71..73
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 75..80
FT /evidence="ECO:0007829|PDB:5B74"
FT TURN 83..85
FT /evidence="ECO:0007829|PDB:4RA9"
FT HELIX 86..96
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 97..99
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 101..107
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 120..131
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 137..142
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 145..148
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 149..151
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 152..154
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 157..160
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 163..165
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 171..175
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 178..181
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 202..206
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 214..222
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 226..234
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 239..243
FT /evidence="ECO:0007829|PDB:4RA9"
FT HELIX 244..251
FT /evidence="ECO:0007829|PDB:5B74"
FT TURN 252..254
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 257..266
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 274..281
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 284..286
FT /evidence="ECO:0007829|PDB:4RA6"
FT HELIX 292..303
FT /evidence="ECO:0007829|PDB:5B74"
FT HELIX 309..316
FT /evidence="ECO:0007829|PDB:5B74"
FT STRAND 320..323
FT /evidence="ECO:0007829|PDB:5B74"
SQ SEQUENCE 326 AA; 35765 MW; 8A9DB1F01F52CA0C CRC64;
MKILLIGMGG TIASVKGENG YEASLSVKEV LDIAGIKDCE DCDFLDLKNV DSTLIQPEDW
VDLAETLYKN VKKYDGIIVT HGTDTLAYTS SMISFMLRNP PIPIVFTGSM IPATEENSDA
PLNLQTAIKF ATSGIRGVYV AFNGKVMLGV RTSKVRTMSR DAFESINYPI IAELRGEDLV
VNFIPKFNNG EVTLDLRHDP KVLVIKLIPG LSGDIFRAAV ELGYRGIVIE GYGAGGIPYR
GSDLLQTIEE LSKEIPIVMT TQAMYDGVDL TRYKVGRLAL RAGVIPAGDM TKEATVTKLM
WILGHTNNVE EIKVLMRKNL VGELRD
