Literature summary for 3.4.11.9 extracted from

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 (2018), Proteins, 87, 212-225 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3) pLysS	Escherichia coli
recombinant expression of wild-type and mutant His-tagged enzyme in Escherichia coli strain BL21(DE3) pLysS	Deinococcus radiodurans

Crystallization (Commentary)

Crystallization (Comment)	Organism
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 crystallization solution containing 0.1 M phospho-citrate, pH 4.6, 0.2 M NaCl, and 22% PEG 8000, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement using the putative prolidase from Telmatobius sibiricus (PDB ID: 4FKC) as template	Deinococcus radiodurans
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	Escherichia coli

Crystallization (Comment)

Organism

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 crystallization solution containing 0.1 M phospho-citrate, pH 4.6, 0.2 M NaCl, and 22% PEG 8000, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement using the putative prolidase from Telmatobius sibiricus (PDB ID: 4FKC) as template

Deinococcus radiodurans

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

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
D53A	site-directed mutagenesis, mutant D53A is significantly less active than the wild-type protein toward Xaa-Pro-Ala tripeptides	Deinococcus radiodurans
H193A	site-directed mutagenesis, inactive mutant, the mutant shows 4fold reduced activity compared to the wild-type	Deinococcus radiodurans
H281A	site-directed mutagenesis, the H281A mutation leads to 10fold reduction in the activity compared to wild-type possibly because of poor substrate binding	Deinococcus radiodurans
R298A	site-directed mutagenesis	Deinococcus radiodurans
R55A	site-directed mutagenesis, mutant R55A is significantly less active than the wild-type protein toward Xaa-Pro-Ala tripeptides	Deinococcus radiodurans
Y56A	site-directed mutagenesis, mutant Y56A is significantly less active than the wild-type protein toward Xaa-Pro-Ala tripeptides	Deinococcus radiodurans

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.15	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
0.23	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
0.46	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
1.9	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
2.1	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
3	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
3.5	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
3.9	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
4	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
6	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
6.1	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
6.3	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
7.9	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
8.4	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
9	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
11.9	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Mycobacterium tuberculosis	5737	-

Metals/Ions

Metals/Ions	Comment	Organism
Mn2+	required	Escherichia coli
Mn2+	required	Deinococcus radiodurans
Mn2+	required	Mycobacterium tuberculosis
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	Escherichia coli
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 of Dr-smAPP is coordinated by O3 of phosphate ion, His285 Nepsilon2, and Glu328 Oepsilon1 in the equatorial plane and Asp221 Odelta2 and Glu314 Oepsilon2 in the axial sites. Metal ZnB of Dr-smAPP is coordinated by O3 of phosphate ion, Asp210 Odelta1, Asp221 Odelta1, and Glu328 Oepsilon2. Glu328 and Asp221 act as bidentate ligands and bind to both the metals	Deinococcus radiodurans

Organism

Organism	UniProt	Comment	Textmining
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	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography, tag cleavage by TEV protease, dialysis, and gel filtration	Escherichia coli
recombinant wild-type and mutant His-tagged enzyme from Escherichia coli by nickel affinity chromatography, tag cleavage by TEV protease, dialysis, and gel filtration	Deinococcus radiodurans

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans R1	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans DSM 20539	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans VKM B-1422	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans NCIMB 9279	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans JCM 16871	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans LMG 4051	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans ATCC 13939	Arg + Pro-Ala	-	?
Arg-Pro-Ala + H2O	-	Deinococcus radiodurans NBRC 15346	Arg + Pro-Ala	-	?
Met-Ala-Ala + H2O	-	Escherichia coli	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Mycobacterium tuberculosis	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans R1	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans DSM 20539	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans VKM B-1422	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Mycobacterium tuberculosis H37Rv	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Mycobacterium tuberculosis ATCC 25618	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans NCIMB 9279	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans JCM 16871	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans LMG 4051	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans ATCC 13939	Met + Ala-Ala	-	?
Met-Ala-Ala + H2O	-	Deinococcus radiodurans NBRC 15346	Met + Ala-Ala	-	?
Met-Pro + H2O	-	Escherichia coli	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans	Met + Pro	-	?
Met-Pro + H2O	-	Mycobacterium tuberculosis	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans R1	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans DSM 20539	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans VKM B-1422	Met + Pro	-	?
Met-Pro + H2O	-	Mycobacterium tuberculosis H37Rv	Met + Pro	-	?
Met-Pro + H2O	-	Mycobacterium tuberculosis ATCC 25618	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans NCIMB 9279	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans JCM 16871	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans LMG 4051	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans ATCC 13939	Met + Pro	-	?
Met-Pro + H2O	-	Deinococcus radiodurans NBRC 15346	Met + Pro	-	?
Met-Pro-Ala + H2O	-	Escherichia coli	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Mycobacterium tuberculosis	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans R1	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans DSM 20539	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans VKM B-1422	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Mycobacterium tuberculosis H37Rv	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Mycobacterium tuberculosis ATCC 25618	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans NCIMB 9279	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans JCM 16871	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans LMG 4051	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans ATCC 13939	Met + Pro-Ala	-	?
Met-Pro-Ala + H2O	-	Deinococcus radiodurans NBRC 15346	Met + Pro-Ala	-	?
additional information	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	Escherichia coli	?	-	?
additional information	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	Deinococcus radiodurans	?	-	?
additional information	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	Mycobacterium tuberculosis	?	-	?
additional information	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	Deinococcus radiodurans R1	?	-	?
additional information	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	Deinococcus radiodurans DSM 20539	?	-	?
additional information	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	Deinococcus radiodurans VKM B-1422	?	-	?
additional information	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	Mycobacterium tuberculosis H37Rv	?	-	?
additional information	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	Mycobacterium tuberculosis ATCC 25618	?	-	?
additional information	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	Deinococcus radiodurans NCIMB 9279	?	-	?
additional information	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	Deinococcus radiodurans JCM 16871	?	-	?
additional information	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	Deinococcus radiodurans LMG 4051	?	-	?
additional information	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	Deinococcus radiodurans ATCC 13939	?	-	?
additional information	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	Deinococcus radiodurans NBRC 15346	?	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 37100, SDS-PAGE	Deinococcus radiodurans
homodimer	2 * 39600, SDS-PAGE	Escherichia coli
homodimer	2 * 38700, SDS-PAGE	Mycobacterium tuberculosis

Synonyms

Synonyms	Comment	Organism
Dr-smAPP	-	Deinococcus radiodurans
Ec-smAPP	-	Escherichia coli
M24B peptidase	-	Escherichia coli
M24B peptidase	-	Deinococcus radiodurans
M24B peptidase	-	Mycobacterium tuberculosis
Mt-smAPP	-	Mycobacterium tuberculosis
PepQ	-	Mycobacterium tuberculosis
peptidase PepQ	-	Mycobacterium tuberculosis
small aminopeptidase-P	-	Escherichia coli
small aminopeptidase-P	-	Deinococcus radiodurans
small aminopeptidase-P	-	Mycobacterium tuberculosis
YpdF	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
55	-	assay at	Escherichia coli
55	-	assay at	Deinococcus radiodurans
55	-	assay at	Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
1.5	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
2	8	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
5	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
10.5	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
18	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
29	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
33	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
35	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
97	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
101	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
126	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
140	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
144	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
152	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
240	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
242	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Escherichia coli
8	-	assay at	Deinococcus radiodurans
8	-	assay at	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the M24B subfamily of aminoproteases	Escherichia coli
evolution	the enzyme belongs to the M24B subfamily of aminoproteases	Deinococcus radiodurans
additional information	the active site includes the DXRY motif. Structure comparisons of small aminopeptidases-P	Mycobacterium tuberculosis
additional information	the protein adopts a two-domain structure typical of the M24B subfamily with an N-terminal domain (residues 1-121) and a C-terminal domain (residues 122-349). The C-terminal domain, adopting a typical pitabread-fold, houses the metal binding active site. Residues Asp53, Arg55, and Tyr56 are part of the conserved DXRY motif, which is important for enzymatic activity, His193 is expected to interact with the DXRY motif in the closed conformation and may also be involved in substrate binding, His281 is expected to be part of the proline binding pocket, and Arg298 is expected to interact with tripeptide and longer peptide substrates. Residue His93 is expected to interact with the DXRY motif in the closed conformation and may also be involved in substrate binding, His281 is expected to be part of the proline binding pocket, and Arg298 is expected to interact with tripeptide and longer peptide substratesBoth His281 and Arg298 residues are found to be disordered in the Dr-smAPP structure. The active site includes the DXRY motif. Structure comparisons of small aminopeptidases-P	Deinococcus radiodurans
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	Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
2.85	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
4.17	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
4.8	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
6.5	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
8.15	-	Met-Pro	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
8.8	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant D53A	Deinococcus radiodurans
9.3	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Escherichia coli
11	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
14	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Mycobacterium tuberculosis
18	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
24	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
29	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant mutant R55A	Deinococcus radiodurans
36	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant mutant Y56A	Deinococcus radiodurans
70	-	Met-Ala-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
114	-	Arg-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans
127	-	Met-Pro-Ala	pH 8.0, 55°C, recombinant wild-type	Deinococcus radiodurans