Limits...
Orthologous and Paralogous AmpD Peptidoglycan Amidases from Gram-Negative Bacteria

View Article: PubMed Central - PubMed

ABSTRACT

Cell wall recycling and β-lactam antibiotic resistance are linked in Enterobacteriaceae and in Pseudomonas aeruginosa. This process involves a large number of murolytic enzymes, among them a cytoplasmic peptidoglycan amidase AmpD, which plays an essential role by cleaving the peptide stem from key intermediates en route to the β-lactamase production (a resistance mechanism) and cell wall recycling. Uniquely, P. aeruginosa has two additional paralogues of AmpD, designated AmpDh2 and AmpDh3, which are periplasmic enzymes. Despite the fact that AmpDh2 and AmpDh3 share a common motif for their respective catalytic domains, they are each comprised of multidomain architectures and exhibit distinct oligomerization properties. We review herein the structural and biochemical properties of orthologous and paralogous AmpD proteins and discuss their implication in cell wall recycling and antibiotic resistance processes.

No MeSH data available.


Three-dimensional structures of the different AmpDs in their monomeric forms. (a) Structural superimposition of AmpD on its inactive (NMR, colored in orange) versus its active state (X-ray, colored in green). (b) Structural superimposition of the active form of cytosolic AmpD (green cartoon), the catalytic domain of periplasmic AmpDh2 (blue cartoon) and the catalytic domain of periplasmic AmpDh3 (pink cartoon). (c) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow. (d) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036320&req=5

f3: Three-dimensional structures of the different AmpDs in their monomeric forms. (a) Structural superimposition of AmpD on its inactive (NMR, colored in orange) versus its active state (X-ray, colored in green). (b) Structural superimposition of the active form of cytosolic AmpD (green cartoon), the catalytic domain of periplasmic AmpDh2 (blue cartoon) and the catalytic domain of periplasmic AmpDh3 (pink cartoon). (c) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow. (d) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow.

Mentions: The reported NMR structure of AmpD from C. freundii (no structural information has been reported for the P. aeruginosa ortholog) revealed, for the first time, similarities with bacteriophage T7 lysozyme and with eukaryotic PGRPs.25 This structure presented a small active site incapable of accommodating its substrate. The crystal structures of AmpD in its apo- and holoenzyme forms at two different pH values, as well as in complex with the reaction products revealed a structure that, while preserving the general fold observed in the NMR structure, exhibited a larger active site around the catalytic zinc ion14 (Fig. 3a). A large rmsd value of 3.9 Å (for all Cα atoms) was calculated from superimposing NMR and crystal structures, indicating that the highest changes were concentrated in four specific regions of the protein (r1–r4) surrounding the catalytic zinc ion (Fig. 3a). Since AmpD crystals in the presence of the substrate showed catalytic activity, the crystal structure conformation was proposed to be the enzyme active form, while the NMR structure was considered to be that of the inactive form by exhibiting a “closed” conformation.14 The reason why this PG amidase would present such activation mechanism is still unknown; however it is likely that in the cytoplasm this enzyme would have fortuitous proteolytic activities that an activation mechanism would moderate.14 In addition, sequence conservation of the regions involved in activation of all bacterial cytosolic AmpD enzymes and in some intracellular PGRP with peptidase activity (but not in periplasmic or extracellular enzymes) further supports this hypothesis.


Orthologous and Paralogous AmpD Peptidoglycan Amidases from Gram-Negative Bacteria
Three-dimensional structures of the different AmpDs in their monomeric forms. (a) Structural superimposition of AmpD on its inactive (NMR, colored in orange) versus its active state (X-ray, colored in green). (b) Structural superimposition of the active form of cytosolic AmpD (green cartoon), the catalytic domain of periplasmic AmpDh2 (blue cartoon) and the catalytic domain of periplasmic AmpDh3 (pink cartoon). (c) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow. (d) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036320&req=5

f3: Three-dimensional structures of the different AmpDs in their monomeric forms. (a) Structural superimposition of AmpD on its inactive (NMR, colored in orange) versus its active state (X-ray, colored in green). (b) Structural superimposition of the active form of cytosolic AmpD (green cartoon), the catalytic domain of periplasmic AmpDh2 (blue cartoon) and the catalytic domain of periplasmic AmpDh3 (pink cartoon). (c) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow. (d) Crystal structure of the monomer of AmpDh2 showing the multidomain arrangement. The N-terminal coiled-coil/loop colored in gray, the catalytic domain colored in blue and the PG-binding domain colored in yellow.
Mentions: The reported NMR structure of AmpD from C. freundii (no structural information has been reported for the P. aeruginosa ortholog) revealed, for the first time, similarities with bacteriophage T7 lysozyme and with eukaryotic PGRPs.25 This structure presented a small active site incapable of accommodating its substrate. The crystal structures of AmpD in its apo- and holoenzyme forms at two different pH values, as well as in complex with the reaction products revealed a structure that, while preserving the general fold observed in the NMR structure, exhibited a larger active site around the catalytic zinc ion14 (Fig. 3a). A large rmsd value of 3.9 Å (for all Cα atoms) was calculated from superimposing NMR and crystal structures, indicating that the highest changes were concentrated in four specific regions of the protein (r1–r4) surrounding the catalytic zinc ion (Fig. 3a). Since AmpD crystals in the presence of the substrate showed catalytic activity, the crystal structure conformation was proposed to be the enzyme active form, while the NMR structure was considered to be that of the inactive form by exhibiting a “closed” conformation.14 The reason why this PG amidase would present such activation mechanism is still unknown; however it is likely that in the cytoplasm this enzyme would have fortuitous proteolytic activities that an activation mechanism would moderate.14 In addition, sequence conservation of the regions involved in activation of all bacterial cytosolic AmpD enzymes and in some intracellular PGRP with peptidase activity (but not in periplasmic or extracellular enzymes) further supports this hypothesis.

View Article: PubMed Central - PubMed

ABSTRACT

Cell wall recycling and β-lactam antibiotic resistance are linked in Enterobacteriaceae and in Pseudomonas aeruginosa. This process involves a large number of murolytic enzymes, among them a cytoplasmic peptidoglycan amidase AmpD, which plays an essential role by cleaving the peptide stem from key intermediates en route to the β-lactamase production (a resistance mechanism) and cell wall recycling. Uniquely, P. aeruginosa has two additional paralogues of AmpD, designated AmpDh2 and AmpDh3, which are periplasmic enzymes. Despite the fact that AmpDh2 and AmpDh3 share a common motif for their respective catalytic domains, they are each comprised of multidomain architectures and exhibit distinct oligomerization properties. We review herein the structural and biochemical properties of orthologous and paralogous AmpD proteins and discuss their implication in cell wall recycling and antibiotic resistance processes.

No MeSH data available.