Literature summary for 3.4.11.9 extracted from

Drinkwater, N.; Sivaraman, K.K.; Bamert, R.S.; Rut, W.; Mohamed, K.; Vinh, N.B.; Scammells, P.J.; Drag, M.; McGowan, S.
Structure and substrate fingerprint of aminopeptidase P from Plasmodium falciparum (2016), Biochem. J., 473, 3189-3204 .

Application

Application	Comment	Organism
drug development	the enzyme PfAPP might be a potential target for an antimalarial therapeutic strategy	Plasmodium falciparum

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)	Plasmodium falciparum

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme PfAPP both unliganded and in complex with inhibitor apstatin, hanging drop vapor diffusion method, mixing of 2:1 vl/v ratio of 6 mg/ml protein solution with reservoir containing 40-50% 2-methyl-2,4-pentanediol, and 0.1 M sodium cacodylate, pH 5.6-6.2, 3-7 days, X-ray diffraction structure determination and analysis at 2.30-2.35 A resolution. Crystals of the apstatin:PfAPP complex are obtained by soaking unliganded crystals in mother liquor supplemented with 2 mM apstatin for 1 h. For the unliganded PfAPP structure, the molecular replacement search model is prepared from the structure of human APP1 (PDB ID 3CTZ)	Plasmodium falciparum

Crystallization (Comment)

Organism

purified recombinant enzyme PfAPP both unliganded and in complex with inhibitor apstatin, hanging drop vapor diffusion method, mixing of 2:1 vl/v ratio of 6 mg/ml protein solution with reservoir containing 40-50% 2-methyl-2,4-pentanediol, and 0.1 M sodium cacodylate, pH 5.6-6.2, 3-7 days, X-ray diffraction structure determination and analysis at 2.30-2.35 A resolution. Crystals of the apstatin:PfAPP complex are obtained by soaking unliganded crystals in mother liquor supplemented with 2 mM apstatin for 1 h. For the unliganded PfAPP structure, the molecular replacement search model is prepared from the structure of human APP1 (PDB ID 3CTZ)

Plasmodium falciparum

Inhibitors

Inhibitors	Comment	Organism	Structure
apstatin	a APP inhibitor	Plasmodium falciparum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mn2+	the active site is internally located at the junction of the three domains and shows a di-metal coordination consistent with the presence of two catalytic manganese ions	Plasmodium falciparum

Organism

Organism	UniProt	Comment	Textmining
Plasmodium falciparum	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration	Plasmodium falciparum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
1480	-	purified recombinant enzyme, pH 8.0, 37°C	Plasmodium falciparum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
FPHFD + H2O	a hemoglobin peptide	Plasmodium falciparum	L-Phe + PHFD	-	?
Gly-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	Gly + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Ala-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Ala + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Arg-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Arg + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Asn-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-ASn + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Asp-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	low activity	Plasmodium falciparum	L-Asp + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Gln-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Gln + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Glu-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Glu + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-His-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-His + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Ile-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Ile + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Leu 7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Leu + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Lys-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Lys + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Met-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Met + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Nle-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Nle + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Phe-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Phe + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Pro-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Pro + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Ser-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Ser + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Thr-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Thr + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Trp-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	best substrate	Plasmodium falciparum	L-Trp + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Tyr-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Tyr + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
L-Val-Pro-7-amido-4-carbamoylmethylcoumarin + H2O	-	Plasmodium falciparum	L-Val + Pro-7-amido-4-carbamoylmethylcoumarin	-	?
additional information	substrate specificity, overview. Design and synthesis of a library composed of 20 fluorogenic substrates, which is used to determine the substrate fingerprint of mature PfAPP (PfAPP, residues 121-777). The enzyme from Plasmodium falciparum can catalyze the removal of any residue immediately prior to a proline. The coupled assay uses a prolyl iminopeptidase (EC 3.4.11.5) to release the free 7-amino-4-carbamoylmethylcoumarin for fluorogenic detection	Plasmodium falciparum	?	-	?
RPPGFSPFR + H2O	i.e. bradykinin	Plasmodium falciparum	L-Arg + PPGFSPFR	-	?
YPWTQ + H2O	a hemoglobin peptide	Plasmodium falciparum	L-Tyr + PWTQ	-	?

Subunits

Subunits	Comment	Organism
homodimer	the enzyme structure shows a homodimer associated by an extensive interface between the catalytic domains (domain III) of both monomers. The active site is internally located at the junction of the three domains and shows a di-metal coordination consistent with the presence of two catalytic manganese ions	Plasmodium falciparum

Synonyms

Synonyms	Comment	Organism
aminopeptidase P	-	Plasmodium falciparum
PfAPP	-	Plasmodium falciparum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Plasmodium falciparum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Plasmodium falciparum

General Information

General Information	Comment	Organism
metabolism	four metalloaminopeptidases (MAPs) play a role in peptide turnover in Pf parasites: leucyl aminopeptidase (PfA-M17), alanyl aminopeptidase (PfA-M1), aspartyl aminopeptidase (PfM18AAP), and aminopeptidase P (PfAPP). The substrate profile shows that PfAPP has the capacity to catalyze the removal of any N-terminal amino acid residue from peptides with a P1' proline, and that the other MAPs in Plasmodium falciparum are unable to perform this function	Plasmodium falciparum
additional information	three-dimensional structure analysis and structure-function analysis, structure comparisons, overview	Plasmodium falciparum
physiological function	peptide recycling, the process by which cellular proteins are broken down to single amino acid residues, is critical to parasite survival. In blood-stage malaria parasites, two major processes are responsible for peptide turnover: proteasomal (within the cytosol) and vacuolar (in the specialized digestive food vacuole). The vacuolar pathway is responsible for the digestion of 60-80% of host cell hemoglobin, which is imported into the digestive vacuole and degraded into free amino acids. This process is absolutely necessary for parasite growth and development. The final step of peptide turnover, the removal of N-terminal amino acids from short polypeptide chains, is catalyzed by a panel of aminopeptidases, which work in concert according to different substrate specificities, to complete protein digestion. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP)	Plasmodium falciparum