title: Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae.
abstract: The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, 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. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release.
authors.affiliation: From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Jonathan D
authors.identifier: http://orcid.org/0000-0002-2637-9720
authors.initials: JD
authors.lastname: Lenz
authors.affiliation: the Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Stohl
authors.affiliation: the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, and.
authors.forename: Rosanna M
authors.initials: RM
authors.lastname: Robertson
authors.affiliation: From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Kathleen T
authors.initials: KT
authors.lastname: Hackett
authors.affiliation: From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Fisher
authors.affiliation: From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Kalia
authors.initials: K
authors.lastname: Xiong
authors.affiliation: the Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana 46556.
authors.forename: Mijoon
authors.initials: M
authors.lastname: Lee
authors.affiliation: the Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana 46556.
authors.forename: Dusan
authors.initials: D
authors.lastname: Hesek
authors.affiliation: the Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana 46556.
authors.forename: Shahriar
authors.initials: S
authors.lastname: Mobashery
authors.affiliation: the Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
authors.forename: H Steven
authors.initials: HS
authors.lastname: Seifert
authors.affiliation: the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Davies
authors.affiliation: From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin 53706, jpdillard@wisc.edu.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Dillard
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Gram-negative bacteria; NOD-like receptor (NLR); Neisseria gonorrhoeae; amidase; cell separation; cell wall; infectious disease; pathogen-associated molecular pattern (PAMP); peptidoglycan