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Information on EC 3.4.11.9 - Xaa-Pro aminopeptidase and Organism(s) Escherichia coli and UniProt Accession P15034
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3.4.11.9 Xaa-Pro aminopeptidaseSpecify your search results
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
release of any N-terminal amino acid, including proline, that is linked to proline, even from a dipeptide or tripeptide
Synonyms
xpnpep2, membrane-bound app, xpnpep3, xpnpep1, appro, tgapp, tvmp50, x-prolyl-dipeptidyl aminopeptidase, aminopeptidase p1, x-prolyl aminopeptidase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
aminoacylproline aminopeptidase
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-
-
-
aminopeptidase P
aminopeptidase, aminoacylproline
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-
-
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AP-P
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-
-
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APP
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-
-
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mAmP
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-
-
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Membrane-bound AmP
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Membrane-bound APP
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-
-
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proline aminopeptidase
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X-Pro aminopeptidase
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-
-
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Xaa-Pro aminopeptidase
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-
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YpdF
aminopeptidase P
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-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
release of any N-terminal amino acid, including proline, that is linked to proline, even from a dipeptide or tripeptide
H243 stabilizes substrate binding, H361 stabilizes substrate binding and the gem-diol catalytic intermediate. H350 forms part of a hydrophobic binding pocket that gives the enzyme its proline specificity
release of any N-terminal amino acid, including proline, that is linked to proline, even from a dipeptide or tripeptide
release of any N-terminal amino acid, including proline, that is linked to proline, even from a dipeptide or tripeptide
mechanism, cis-trans specificity
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release of any N-terminal amino acid, including proline, that is linked to proline, even from a dipeptide or tripeptide
R404 participates in proton relay and in the hydrogen bond network
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CAS REGISTRY NUMBER
COMMENTARY
37288-66-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
Abz-L-Lys-L-Pro-L-Pro-4-nitroanilide + H2O
Abz-L-Lys + L-Pro-L-Pro-4-nitroanilide
-
-
-
?
(4-nitro)Phe-Pro-HN-CH2-CH2-NH-o-aminobenzoyl + H2O
(4-nitro)Phe + Pro-HN-CH2-CH2-NH-o-aminobenzoyl
-
-
-
?
(4-nitro)Phe-Pro-Pro-HN-CH2-CH2-NH-o-aminobenzoyl + H2O
(4-nitro)Phe + Pro-Pro-HN-CH2-CH2-NH-o-aminobenzoyl
-
-
-
?
Abz-L-Lys-L-Pro-L-Pro-p-nitroanilide + H2O
Abz-L-Lys + L-Pro-L-Pro-p-nitroanilide
-
-
-
-
?
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg + H2O
Arg + Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
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i.e. bradykinin
one Arg is released per mol of bradykinin in less than 5 min, the following Pro residue is released within 1 h
?
centrosomal protein 290 kDa/NPHP6 + H2O
?
-
ciliary proteome is screened for proteins with a proline in the second position: 3 candidate substrates centrosomal protein 290 kDa/NPHP6 (CEP290/NPHP6), Alstrom syndrome 1 (ALMS1), and leucine rich repeat containing 50 (LRRC50), known to cause cystic renal disease are shown to be cleaved by ecAPP
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-
?
Gly-Pro-4-methylcoumarin 7-amide + H2O
Gly + Pro-4-methylcoumarin 7-amide
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-
-
-
?
L-Asn-L-Pro-L-Thr-L-Asn-L-Leu-L-His + H2O
L-Asn + L-Pro-L-Thr-L-Asn-L-Leu-L-His
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-
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-
?
leucine rich repeat containing 50 + H2O
?
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ciliary proteome is screened for proteins with a proline in the second position: 3 candidate substrates centrosomal protein 290 kDa/NPHP6 (CEP290/NPHP6), Alstrom syndrome 1 (ALMS1), and leucine rich repeat containing 50 (LRRC50), known to cause cystic renal disease are shown to be cleaved by ecAPP
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-
?
papain + H2O
?
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reduced and carboxymethylated, with the N-terminal sequence Ile-Pro-Glu-Tyr-Val
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?
additional information
?
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peptides in which L-Pro is replaced by N-methyl-L-Ala or L-Ala are extremely poor substrates
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?
additional information
?
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peptides in which L-Pro is replaced by N-methyl-L-Ala or L-Ala are extremely poor substrates
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-
?
additional information
?
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the enzyme can only hydrolyze the trans form of the X-L-Pro-peptide bond, the cis form has to isomerize before it can be cleaved
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-
?
additional information
?
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enzyme hydrolyzes the Xaa-Pro peptide bond when the first amino acid is Asn, Ala, or Met
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-
?
additional information
?
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ciliary proteome is screened for proteins with a proline in the second position: 3 candidate substrates centrosomal protein 290 kDa/NPHP6 (CEP290/NPHP6), Alstrom syndrome 1 (ALMS1), and leucine rich repeat containing 50 (LRRC50), known to cause cystic renal disease are shown to be cleaved by ecAPP
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-
?
additional information
?
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substrate specificity for Xaa-Pro dipeptides and Xaa-Pro-Ala tripeptides is analyzed, the enzyme is significantly more active toward the tripeptides than the corresponding dipeptides
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-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mn2+
Co2+
Mn2+
Zn2+
required, di-metal center, one metal ion (ZnA) is penta-coordinated and exhibits distorted trigonal bipyramidal geometry, whereas the other (ZnB) is tetra-coordinated and exhibits a tetrahedral geometry. Metal ZnA is coordinated by O1 of cacodylate ion, Glu335 Oepsilon2, Glu321 Oepsilon2, His292 Nepsilon2, and Asp223 Odelta2. Metal ZnB is coordinated by O1 of cacodylate ion, Glu335 Oepsilon1, Asp212 Odelta1, and Asp223 Odelta1. Glu335 and Asp223 act as bidentate ligands and bind to both the metals
additional information
Mn2+
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wild-type and mutat R404A, binding of Mn2+ with a stoichiometry of 2 per monomer
additional information
-
the active site contains a dinuclear metal binding site, the enzyme contains 12 metal atoms per molecule, 2 of which are Mn2+ ions
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
L-Pro-L-Leu
product inhibition, a third metal binding site is formed by two conserved His-residues and L-Pro-L-Leu
acetyl-Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-2-aminobenzoyl
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0.5 mM, 30% inhibition of hydrolysis of (4-nitr)Phe-Pro-Pro-HN-CH2-CH2-NH-o-aminobenzoyl
apstatin
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binds to the active site with its N-terminal amino group coordinated to one of the two Mn(II) ions at the metal center
Pro-HN-CH2-CH2-NH-2-aminobenzoyl
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0.01 mM, complete inhibition of hydrolysis of (4-nitro)Phe-Pro-Pro-HN-CH2-CH2-NH-2-aminobenzoyl
additional information
not inhibitory: L-Ala-(N-methyl)-L-Ala-L-Ala, L-Ala-L-Ala-L-Ala
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additional information
-
not inhibitory: L-Ala-(N-methyl)-L-Ala-L-Ala, L-Ala-L-Ala-L-Ala
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.087
2-aminobenzoyl-L-Lys-L-Pro-L-Pro-4-nitroanilide
wild-type, pH 8.1, 37°C
0.14
2-aminobenzoyl-L-Lys-L-Pro-L-Pro-4-nitroanilide
mutant H350A, pH 8.1, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0603
(2R,3R)-3-amino-1-oxo-4-phenyl-1-(1,3-thiazolidin-3-yl)butan-2-ol
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pH 7.4, 30°C
0.0282
(2R,3S)-3-amino-1-oxo-4-phenyl-1-(1,3-thiazolidin-3-yl)butan-2-ol
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pH 7.4, 30°C
0.0307
(2S,3R)-3-amino-1-oxo-4-phenyl-1-(1,3-thiazolidin-3-yl)butan-2-ol
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pH 7.4, 30°C
0.0198
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl pyrrolidide
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pH 7.4, 30°C
0.00126
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-Pro-L-Phe-OMe
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pH 7.4, 30°C
0.014
(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-thiazolidide
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pH 7.4, 30°C
0.0229
(2S,3R)-3-amino-5-methyl-1-oxo-1-(1,3-thiazolidin-3-yl)hexan-2-ol
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pH 7.4, 30°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
55
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
the protein adopts a two-domain structure typical of the M24B subfamily with an N-terminal domain (residues 1-123) and a C-terminal domain (residues 124-360). The C-terminal domain, adopting a typical pita-bread-fold, houses the metal binding active site. The active site includes the DXRY motif. Structure comparisons of small aminopeptidases-P
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexamer
-
arranged in 2 types of tetramers, one tetramer comprises 4 crystallographically independent subunits, while the other tetramer comprises 2 pairs of subunits related by a crystallographic 2fold axis
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Mn(II)-form of enzyme and substituted with Mg2+, Zn2+, Ca2+, Na+ and apo-enzyme in complex with L-Val-L-Pro-L-Leu
mutants E383A, H361A and H243A in complex with substrate L-Val-L-Pro-L-Leu. Substrate interacts with one of the active site metal ions via its terminal amino group
8 mg/ml purified recombinant enzyme in Tris, pH 8.5, hanging drop vapour diffusion method, 0.003 ml mixed with 0.002 ml reservoir solution containing 25% PEG 4000, 0.1 M Tris-HCl, pH 8.0, 0.2 M sodium acetate, and 1 mM MnCl2, 1 month at 4°C, cryoprotectant is 2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis at 2.4 A resolution, modeling
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purified recombinant enzyme, mixing of 0.002 ml of about 15 mg/ml protein in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, with 0.002 ml of crystallization solution containing 1.4 M trisodium citrate, 0.1 M sodium cacodylate, pH 6.5, 10% glycerol, and 0.5 mM ZnCl2, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using the putative prolidase from Telmatobius sibiricus (PDB ID: 4FKC) as template
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D260A
D271A
E383A
H243A
H350A
H354A
H361A
additional information
-
partial or complete deletion of N-terminal domain, complete loss of enzymic activity
D260A
MnA is bound at the active site, but the MnB site is empty. Loss of catalytic activity
D271A
no atoms of Mn(II) are bound at the active site. Loss of catalytic activity
E383A
both Mn-(II) atoms are present in the active site, but the bridging solvent molecule is located significantly farther from the metals than in wild-type. Loss of catalytic activity
E383A
crystallization data. One of the two metal sites is only partially occupied
H243A
crystallization data. Mutant is capable of binding the substrate L-Val-L-Pro-L-Leu
H350A
loss of catalytic activity. H350 forms part of a hydrophobic binding pocket that gives the enzym its proline specificity
H354A
MnB is present at the active site at less than full occupancy, and a water molecule occupies the MnA site. Loss of catalytic activity
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8 - 9
8
-
50°C, 30 min, 50% loss of activity of enzyme form APP-I, 60°C, 50% loss of activity of enzyme form APP-II
36039
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography, tag cleavage by TEV protease, dialysis, and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3) pLysS
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yoshimoto, T.; Murayama, N.; Tsuru, D.
A novel assay method for aminopeptidase P and partial purification of two types of the enzyme in Escherichia coli
Agric. Biol. Chem.
52
1957-1963
1988
Escherichia coli
-
Yoshimoto, T.; Murayama, N.; Honda, T.; Tone, H.; Tsuru, D.
Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme
J. Biochem.
104
93-97
1988
Escherichia coli
Lin, L.N.; Brandts, J.F.
Kinetic mechanism for conformational transitions between poly-L-prolines I and II: a study utilizing the cis-trans specificity of a proline-specific protease
Biochemistry
19
3055-3059
1980
Escherichia coli, Escherichia coli B / ATCC 11303
Yaron, A.; Mlynar, D.
Aminopeptidase-P
Biochem. Biophys. Res. Commun.
32
658-663
1968
Escherichia coli, Escherichia coli B / ATCC 11303
Fleminger, G.; Carmel, A.; Goldenberg, D.; Yaron, A.
Fluorogenic substrates for bacterial aminopeptidase P and its analogs detected in human serum and calf lung
Eur. J. Biochem.
125
609-615
1982
Bos taurus, Escherichia coli, Homo sapiens
Lin, L.N.; Brandts, J.F.
Role of cis-trans isomerism of the peptide bond in protease specificity. Kinetic studies on small proline-containing peptides and on polyproline
Biochemistry
18
5037-5042
1979
Escherichia coli
Stckel, A.; Stiebitz, B.; Neubert, K.
Specific inhibitors of aminopeptidase P. Peptides and pseudopeptides of 2-hydroxy-3-amino acids
Adv. Exp. Med. Biol.
421
31-35
1997
Escherichia coli, Rattus norvegicus
Graham, S.C.; Lee, M.; Freeman, H.C.; Guss, J.M.
An orthorhombic form of Escherichia coli aminopeptidase P at 2.4 A resolution
Acta Crystallogr. Sect. D
59
897-902
2003
Escherichia coli
Graham, S.C.; Maher, M.J.; Simmons, W.H.; Freeman, H.C.; Guss, J.M.
Structure of Escherichia coli aminopeptidase P in complex with the inhibitor apstatin
Acta Crystallogr. Sect. D
D60
1770-1779
2004
Escherichia coli
Graham, S.C.; Bond, C.S.; Freeman, H.C.; Guss, J.M.
Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center
Biochemistry
44
13820-13836
2005
Escherichia coli (P15034), Escherichia coli
Jao, S.C.; Huang, L.F.; Hwang, S.M.; Li, W.S.
Tyrosine 387 and arginine 404 are critical in the hydrolytic mechanism of Escherichia coli aminopeptidase P
Biochemistry
45
1547-1553
2006
Escherichia coli
Graham, S.C.; Lilley, P.E.; Lee, M.; Schaeffer, P.M.; Kralicek, A.V.; Dixon, N.E.; Guss, J.M.
Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis
Biochemistry
45
964-975
2006
Escherichia coli (P15034), Escherichia coli
Stockel-Maschek, A.; Stiebitz, B.; Koelsch, R.; Neubert, K.
Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors
Bioorg. Med. Chem.
13
4806-4820
2005
Escherichia coli, Rattus norvegicus
Zheng, Y.; Roberts, R.J.; Kasif, S.; Guan, C.
Characterization of two new aminopeptidases in Escherichia coli
J. Bacteriol.
187
3671-3677
2005
Escherichia coli
Graham, S.C.; Guss, J.M.
Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu
Arch. Biochem. Biophys.
469
200-208
2008
Escherichia coli (P15034), Escherichia coli
OToole, J.F.; Liu, Y.; Davis, E.E.; Westlake, C.J.; Attanasio, M.; Otto, E.A.; Seelow, D.; Nurnberg, G.; Becker, C.; Nuutinen, M.; Kaerppae, M.; Ignatius, J.; Uusimaa, J.; Pakanen, S.; Jaakkola, E.; van den Heuvel, L.P.; Fehrenbach, H.; Wiggins, R.; Goyal, M.; Zhou, W.; Wolf, M.T.; Wise, E.; Helou, J.; A, A.l.
Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy
J. Clin. Invest.
120
791-802
2010
Danio rerio, Escherichia coli, Homo sapiens (Q9NQH7), Homo sapiens
Are, V.; Kumar, A.; Goyal, V.; Gotad, S.; Ghosh, B.; Gadre, R.; Jamdar, S.; Makde, R.
Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases
Proteins
87
212-225
2018
Deinococcus radiodurans (Q9RUY4), Deinococcus radiodurans ATCC 13939 (Q9RUY4), Deinococcus radiodurans DSM 20539 (Q9RUY4), Deinococcus radiodurans JCM 16871 (Q9RUY4), Deinococcus radiodurans LMG 4051 (Q9RUY4), Deinococcus radiodurans NBRC 15346 (Q9RUY4), Deinococcus radiodurans NCIMB 9279 (Q9RUY4), Deinococcus radiodurans R1 (Q9RUY4), Deinococcus radiodurans VKM B-1422 (Q9RUY4), Escherichia coli (P76524), Mycobacterium tuberculosis (I6YDN6), Mycobacterium tuberculosis ATCC 25618 (I6YDN6), Mycobacterium tuberculosis H37Rv (I6YDN6)
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