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Literature summary extracted from

  • Lenz, J.D.; Stohl, E.A.; Robertson, R.M.; Hackett, K.T.; Fisher, K.; Xiong, K.; Lee, M.; Hesek, D.; Mobashery, S.; Seifert, H.S.; Davies, C.; Dillard, J.P.
    Amidase activity of AmiC controls cell separation and stem peptide release and is enhanced by NlpD in Neisseria gonorrhoeae (2016), J. Biol. Chem., 291, 10916-10933 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.5.1.28 NlpD the presence of NlpD enhances AmiC activity Neisseria gonorrhoeae

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.28 gene amiC, sequence comparisons, recombinant expression of C-terminally HA-tagged or His6-tagged wild-type and mutant enzymes in Escherichia coli strain DH5alpha Neisseria gonorrhoeae

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.28 E229D mutation alters peptidoglycan fragment release and causes a defect in cell separation Neisseria gonorrhoeae
3.5.1.28 E229D site-directed mutagenesis, the mutation leads to reduced enzyme activity and causes a defect in cell separation. The amiCE229D mutant releases larger [3H]glucosamine-labeled peptidoglycan fragments relative to the wild-type and no disaccharide. The amiCE229D mutant does not release some [3H]DAP-labeled peptide fragments, similar to a DELTAamiC strain Neisseria gonorrhoeae
3.5.1.28 additional information generation of an enzyme deletion mutant DELTAamiC Neisseria gonorrhoeae
3.5.1.28 Q316K mutation results in an AmiC with increased enzymatic activity on macromolecular peptidoglycan and on the synthetic peptidoglycan derivative GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide). The same mutation also results in cell separation and peptidoglycan fragment release defects Neisseria gonorrhoeae
3.5.1.28 Q316K site-directed mutagenesis, mutation Q316K results in an AmiC with increased enzymatic activity on macromolecular peptidoglycan (PG) and on the synthetic PG derivative. The same Q316K mutation that increases AmiC activity also results in cell separation and PG fragment release defects. But amiCQ316K mutation has only intermediate effects on PG fragment release and causes a slight defect in cell separation. In addition, the mutation amiCQ316K causes increased activity on whole sacculi and lysis of Escherichia coli cells Neisseria gonorrhoeae

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.28 1,10-phenanthroline complete inhibition; enzyme results in complete loss of activity, when dialyzed overnight against the metal chelator Neisseria gonorrhoeae
3.5.1.28 CDTA about 90% inhibition; enzyme results in about 90% loss of activity, when dialyzed overnight against the metal chelator. Some restoration of activity is observed with all of the metal salts (10 mM ZnCl2, MgCl2, CaCl2, CoCl2, or NiCl). The addition of ZnCl2, MgCl2, and CaCl2 is the most effective, restoring 70-80% of the activity, with NiCl2 restoring about 50% and CoCl2 the least effective at only about 25% Neisseria gonorrhoeae
3.5.1.28 EDTA about 70% inhibition; enzyme results in about 70% loss of activity, when dialyzed overnight against the metal chelator Neisseria gonorrhoeae
3.5.1.28 EGTA about 90% inhibition; enzyme results in about 90% loss of activity, when dialyzed overnight against the metal chelator Neisseria gonorrhoeae
3.5.1.28 additional information enzyme residue Gln316 has an autoinhibitory role Neisseria gonorrhoeae

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.5.1.28 periplasm
-
 Neisseria gonorrhoeae
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.1.28 Ca2+ can partially substitute for Zn2+, can restore 70-80% of the activity when added exogenously to the apoenzyme Neisseria gonorrhoeae
3.5.1.28 Co2+ can partially substitute for Zn2+, can restore 25% of the activity when added exogenously to the apoenzyme Neisseria gonorrhoeae
3.5.1.28 Mg2+ can partially substitute for Zn2+, can restore 70-80% of the activity when added exogenously to the apoenzyme Neisseria gonorrhoeae
3.5.1.28 additional information gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments Neisseria gonorrhoeae
3.5.1.28 Ni2+ can partially substitute for Zn2+, can restore 50% of the activity when added exogenously to the apoenzyme Neisseria gonorrhoeae
3.5.1.28 Zn2+ AmiC is a zinc-dependent amidase, but the requirement for the divalent metal ion by this protein is not strict. The zinc ion in the catalytic site is coordinated by five residues: His196, Glu211, His265, Asp267, and Gln299. Zn2+ can restore 70-80% of the activity when added exogenously to the apoenzyme Neisseria gonorrhoeae
3.5.1.28 Zn2+ zinc-dependent amidase, the requirement for the divalent metal ion by this protein is not strict Neisseria gonorrhoeae

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.28 additional information Neisseria gonorrhoeae enzyme AmiC displays high basal activity on peptidoglycan. Gonococcal AmiC (GC-AmiC) activity on whole GC sacculi, solubilization of whole gonococcal sacculi ?
-
 ?
3.5.1.28 additional information Neisseria gonorrhoeae MS11 enzyme AmiC displays high basal activity on peptidoglycan. Gonococcal AmiC (GC-AmiC) activity on whole GC sacculi, solubilization of whole gonococcal sacculi ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.28 Neisseria gonorrhoeae
-
-
-
3.5.1.28 Neisseria gonorrhoeae MS11
-
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
3.5.1.28 proteolytic modification AmiC functions as an autolysin Neisseria gonorrhoeae

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.28
-
 Neisseria gonorrhoeae
3.5.1.28 recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, dialysis, and tag cleavage via TEV protease, followed by another step of nickel affinity chromatography Neisseria gonorrhoeae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.28 GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc + H2O the cell separation amidase can utilize the small synthetic peptidoglycan a pentapeptide fragment as substrate. AmiC can cleave one peptide stem from synthetic PG dimer Neisseria gonorrhoeae ?
-
 ?
3.5.1.28 GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc + H2O the cell separation amidase can utilize the small synthetic peptidoglycan a pentapeptide fragment as substrate. AmiC can cleave one peptide stem from synthetic PG dimer Neisseria gonorrhoeae MS11 ?
-
 ?
3.5.1.28 GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide) + H2O small synthetic peptidoglycan fragment Neisseria gonorrhoeae ?
-
 ?
3.5.1.28 GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide) + H2O small synthetic peptidoglycan fragment Neisseria gonorrhoeae MS11 ?
-
 ?
3.5.1.28 additional information enzyme AmiC displays high basal activity on peptidoglycan. Gonococcal AmiC (GC-AmiC) activity on whole GC sacculi, solubilization of whole gonococcal sacculi Neisseria gonorrhoeae ?
-
 ?
3.5.1.28 additional information enzyme AmiC displays high basal activity on peptidoglycan. Gonococcal AmiC (GC-AmiC) activity on whole GC sacculi, solubilization of whole gonococcal sacculi Neisseria gonorrhoeae MS11 ?
-
 ?
3.5.1.28 peptidoglycan + H2O the enzyme can act on macromolecular peptidoglycan to liberate cross-linked and non-cross-linked peptides Neisseria gonorrhoeae ?
-
 ?
3.5.1.28 peptidoglycan + H2O the enzyme can act on macromolecular peptidoglycan to liberate cross-linked and non-cross-linked peptides Neisseria gonorrhoeae MS11 ?
-
 ?
3.5.1.28 peptidoglycan + H2O enzyme AmiC displays high basal activity on peptidoglycan. In vivo assay of HA-tagged recombinant enzyme in Escherichia coli cells, usage of fluorescein-labeled peptidoglycan. Digesting gonococcal PG with AmiC results in the recovery of soluble fragments of predicted masses matching tripeptide stem (L-Ala-gamma-D-Glu-mDAP), tetrapeptide stem (L-Ala-gamma-D-Glu-mDAP-D-Ala), pentapeptide stem (L-Ala-gamma-D-Glu-mDAPD-Ala-D-Ala), cross-linked tetrapeptide-tripeptide (4-3), crosslinked tetrapeptide-tetrapeptide (4-4), and cross-linked tetrapeptide-tetrapeptide-tetrapeptide (4-4-4). Substrate specificity of wild-type and mutant enzymes and product release analysis, detailed overview Neisseria gonorrhoeae peptidoglycan-derived fragment peptides
-
 ir
3.5.1.28 peptidoglycan + H2O enzyme AmiC displays high basal activity on peptidoglycan. In vivo assay of HA-tagged recombinant enzyme in Escherichia coli cells, usage of fluorescein-labeled peptidoglycan. Digesting gonococcal PG with AmiC results in the recovery of soluble fragments of predicted masses matching tripeptide stem (L-Ala-gamma-D-Glu-mDAP), tetrapeptide stem (L-Ala-gamma-D-Glu-mDAP-D-Ala), pentapeptide stem (L-Ala-gamma-D-Glu-mDAPD-Ala-D-Ala), cross-linked tetrapeptide-tripeptide (4-3), crosslinked tetrapeptide-tetrapeptide (4-4), and cross-linked tetrapeptide-tetrapeptide-tetrapeptide (4-4-4). Substrate specificity of wild-type and mutant enzymes and product release analysis, detailed overview Neisseria gonorrhoeae MS11 peptidoglycan-derived fragment peptides
-
 ir

Synonyms

EC Number Synonyms Comment Organism
3.5.1.28 AmiC
-
 Neisseria gonorrhoeae
3.5.1.28 cell separation amidase
-
 Neisseria gonorrhoeae
3.5.1.28 GC-AmiC
-
 Neisseria gonorrhoeae

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.28 22 37 recombinant enzyme, assay at Neisseria gonorrhoeae

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.28 6 7.5 recombinant enzyme, assay at Neisseria gonorrhoeae

General Information

EC Number General Information Comment Organism
3.5.1.28 evolution AmiC and AmiA structure comparisons. Neisseria gonorrhoeae has only one cell separation amidase (AmiC) in contrast to other baceria, e.g. Escherichia coli Neisseria gonorrhoeae
3.5.1.28 malfunction deletion of amiC from Neisseria gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release. Mutation Q316K results in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Mutation Q316K also results in cell separation and PG fragment release defects Neisseria gonorrhoeae
3.5.1.28 malfunction deletion of amiC from Neisseria gonorrhoeae results in severely impaired cell separation and altered peptidoglycan fragment release Neisseria gonorrhoeae
3.5.1.28 metabolism gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Deletion of amiC results in cells that grow in clusters and have formed septa but fail to separate. This phenotype is similar to that observed in Neisseria gonorrhoeae lacking the lytic transglycosylase LtgC, implying that LtgC may act in concert with AmiC. The amiCE229D mutant releases larger [3H]glucosamine-labeled peptidoglycan fragments, relative to the wild-type, and no disaccharide, overview. Loss of released peptides does not impact NOD-dependent NF-kappaB activation by gonococci Neisseria gonorrhoeae
3.5.1.28 additional information enzyme residue Glu229 is critical for both normal cell separation and the release of peptidoglycan (PG) fragments by gonococci during growth. Residue Gln316 has an autoinhibitory role. AmiC and AmiA structure comparisons. Enzyme residue Glu229 is a critical residue necessary for the enzymatic activity of AmiC Neisseria gonorrhoeae
3.5.1.28 physiological function enzyme AmiC is critical for proper cell separation, is necessary for normal peptidoglycan (PG) fragment release, and functions as an autolysin. The amidase activity of AmiC controls cell separation and stem peptide release and is enhanced by NlpD in Neisseria gonorrhoeae. The activation state is not the only factor determining normal AmiC activity Neisseria gonorrhoeae
3.5.1.28 physiological function the periplasmic N-acetylmuramyl-L-alanine amidase, AmiC, is involved in cell separation Neisseria gonorrhoeae