Pseudomonas aeruginosa LD-Carboxypeptidase, a Serine Peptidase with a Ser-His-Glu Triad and a Nucleophilic Elbow*

Henryk J Korza,Matthias Bochtler

doi:10.1074/jbc.m506328200

Abstract

LD-Carboxypeptidases (EC 3.4.17.13) are named for their ability to cleave amide bonds between l- and d-amino acids, which occur naturally in bacterial peptidoglycan. They are specific for the link between meso-diaminopimelic acid and d-alanine and therefore degrade GlcNAc-MurNAc tetrapeptides to the corresponding tripeptides. As only the tripeptides can be reused as peptidoglycan building blocks, ld-carboxypeptidases are thought to play a role in peptidoglycan recycling. Despite the pharmaceutical interest in peptidoglycan biosynthesis, the fold and catalytic type of ld-carboxypeptidases are unknown. Here, we show that a previously uncharacterized open reading frame in Pseudomonas aeruginosa has ld-carboxypeptidase activity and present the crystal structure of this enzyme. The structure shows that the enzyme consists of an N-terminal beta-sheet and a C-terminal beta-barrel domain. At the interface of the two domains, Ser(115) adopts a highly strained conformation in the context of a strand-turn-helix motif that is similar to the "nucleophilic elbow" in alphabeta-hydrolases. Ser(115) is hydrogen-bonded to a histidine residue, which is oriented by a glutamate residue. All three residues, which occur in the order Ser-Glu-His in the amino acid sequence, are strictly conserved in naturally occurring ld-carboxypeptidases and cannot be mutated to alanines without loss of activity. We conclude that ld-carboxypeptidases are serine peptidases with Ser-His-Glu catalytic triads.

Highlights

Reconverted into peptidoglycan building blocks by the attachment of preformed D-Ala-D-Ala dipeptides
Ursinus et al [2] reported the purification of a norcardicin A-sensitive and thienamycin-insensitive activity, which was originally ascribed to a 43-kDa dimer-forming protein in the periplasm
LD-Carboxypeptidase is sensitive to norcardicin A, but biochemical results suggest that the interaction is noncovalent and does not involve an opening of the lactam ring of the antibiotic [2]

Summary

EXPERIMENTAL PROCEDURES

Cloning—The gene for LD-carboxypeptidase from P. aeruginosa strain PA01 was amplified by standard PCR methods from genomic DNA, adding EcoRI and BamHI sites for cloning. ␣-amylase (Fluka) for 2 h at 37 °C to degrade high molecular mass glycogen and incubated with 240 ␮g of Pronase (Fluka) for 1.5 h at 60 °C to release covalently bound lipoprotein After another wash with boiling 8% SDS, the pellet was resuspended in 1.2 ml of 10 mM Tris (pH 7) and treated overnight with 200 –300 ␮g of lysozyme at 37 °C. Activity Assay—2 ␮g of GlcNAc-MurNAc tetrapeptide (ϳ2 nmol) was incubated with 20 ng of LD-carboxypeptidase (0.57 pmol of protomer) or control for 20 min at 37 °C in either 10 mM Tris (pH 7.0) or crystallization buffer (50 mM citric acid/citrate (pH 4.5)). Plate-like crystals were obtained by vapor diffusion at room temperature (21 °C) when a 6 mg/ml protein solution was mixed in 1:1 ratio with reservoir buffer containing 50 mM citric acid (pH 4.5).

Derivative Selenomethionine

RESULTS

TABLE TWO

DISCUSSION