Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Jonathan D Lenz,Elizabeth A Stohl,Rosanna M Robertson,Kathleen T Hackett,Kathryn Fisher,Kalia Xiong,Mijoon Lee,Dusan Hesek,Shahriar Mobashery,H Steven Seifert,Christopher Davies,Joseph P Dillard

doi:10.1074/jbc.m116.715573

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.

Highlights

IntroductionNeisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea
Neisseria gonorrhoeae is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea
Rather than the peptides and disaccharides released by E. coli, GC release a larger proportion of N-acetylglucosaminyl-1,6-anhydromuramyl-tripeptide and -tetrapeptide fragments, as well as stem peptides, GlcNAc-anhMurNAc disaccharides, and some larger fragments (8 –11)

Summary

Introduction

Neisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea. AmiA and AmiC travel similar paths to the periplasm, it is AmiB and AmiC that contain N-terminal extensions targeting them to the E. coli septum, whereas AmiA remains diffusely distributed in the periplasm [20] The structure of this N-terminal extension (AMIN domain) has been solved and shown to bind peptidoglycan independently of the C-terminal catalytic domain [21]. In E. coli, the LytM homologs EnvC and NlpD lack two of the four predicted metalloendopeptidase active-site residues [25], display no detectable enzymatic activity, and have been shown to act in a regulatory role, enhancing the activity of amidases in specific cognate pairs (AmiA and AmiB with EnvC and AmiC with NlpD) [26, 29]. By purifying GC-AmiC, we were able to define the products of GC-AmiC activity on whole GC sacculi and synthetic PG GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc (pentapeptide) substrate

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Conclusion