Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism

Abstract

obtained by irradiating the crystals for 10 min. with a blue laser (508 nm). Diffraction data were collected at the PXII beamline of the Swiss Light Source at 100K using a MarResearch CCDC detector. The structures were solved in space group P2 1 2 1 2 1 by molecular replacement using the monomer of Dronpa (pdb code 2z1o) as search model. The asymmetric unit contains 12 monomers, associated in three tetramers. The structures were refined at an effective resolution of 3.0 Å for the on-state and 3.15 Å for the off-state. R/R free values converged at 21.1/25.7% for the on-state and 21.3/25.0% for the off-state. PDM1-4 exhibits a β-barrel structure typical for GFP-like proteins. Furthermore, a light-driven cis-trans isomerization of the chromophore is observed. From the structures of the on-and off-states we elucidate that the presence of nickel ions, interacting with His 194 and His 212, decreases the flexibility of the β-strands, resulting in the slower switching kinetics of PDM1-4. The photoswitching mechanism of reversible photoswitchable fluorescent proteins not only arises from the flexibility of the chromophore accompanied by a rearrangement of the proximate residues, but is also influenced by the flexibility of the β-strands.

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage

“…The number of glycines may vary from four to six, or one glycine may be substituted by a serine or an alanine (43,44). Although structures of several bacterial amidases have been determined (19,(45)(46)(47)(48)(49)(50)(51)(52), the amidase AmiD from E. coli is the only catalytically active amidase for which structural data of an uncleaved ligand-enzyme complex have been available prior to this work (21).…”

Bacteriolyses of Bacterial Cell Walls by Cu(II) and Zn(II) Ions Based on Antibacterial Results of Dilution Medium Method and Halo Antibacterial Test

“…The other, zinc ions were accumulated in E. coli periplasmic space, in which the zinc ions are spent to the activation of bacteriolysis of the cell wall. Zinc depending PGN autolysin, amidase PGRPs [41], zinc metallo enzymes AmiD [42], zinc-containing amidase; AmpD [43], zinc-present PGLYRPs [44] . The appearance of the highest antibacterial activity is found to be the zinc sulfate solution.…”

Involvement of Mutation in ampD I, mrcA , and at Least One Additional Gene in β-Lactamase Hyperproduction in Stenotrophomonas maltophilia

“…Furthermore, because loss of AmpDI causes ␤-lactamase hyperproduction in S. maltophilia (14), S. maltophilia AmpR is probably activated by an AHM-peptide that accumulates upon deletion of ampD I. Another alternative would be that a G-AHM-peptide is the AmpR activatory ligand in S. maltophilia, and it is true that we have not confirmed the specificity of S. maltophilia AmpDI for AHM-peptides over G-AHM-peptides, a defining feature of enterobacterial AmpD proteins (30)(31)(32). However, the observation that deletion of nagZ in S. maltophilia abolishes ␤-lactamase hyperproduction caused by deletion of ampD I (33) provides strong evidence against the possibility that G-AHM-peptides are AmpR activatory ligands in S. maltophilia and strengthens the evidence in favor of AHM-peptides fulfilling this role.…”

“…This deleted amino acid sequence, PTLMVARKRD, contains the RKxD quadrad, equivalent to positions 161 to 164 of C. freundii AmpD, which is critical for substrate binding and zinc coordination (30). Accordingly, we had strong reason to suspect that this S. maltophilia AmpDI mutant would be functionless, and this was confirmed experimentally.…”