Crystal Structure of the Actinomadura R39 DD-peptidase Reveals New Domains in Penicillin-binding Proteins

Abstract

Actinomadura sp. R39 produces an exocellular DDpeptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high ␤-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k 2 /K ‫؍‬ 300 mM ؊1 s ؊1 ). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 Å by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded ␤-sheet with two helices on one side, and the other domain is a double threestranded ␤-sheet inserted in the previous domain. Additionally, the 2.4-Å structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the ␤-lactams.

Crystal Structures of Complexes of Bacterial dd-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle

“…24 The solubilized construct is inhibited by β-lactams and also catalyzes the hydrolysis and aminolysis of small D-alanyl peptides and esters, 25 although much less efficiently than the R39 enzyme. The general organization of catalytic functional groups in PBP5 is very similar to that in the R39 DD-peptidase, and both resemble that of a class A β-lactamase; 16 neither, however, contains an analogue of Glu166 of the β-lactamase-the β-lactam deacylation catalyst. Extensive structural details of the low-molecular-mass PBPs are provided in a recent review.…”

“…16 The environment is very similar for monomers A and D, and for monomers B and C, but is different for both pairs.…”

“…14,15 Crystal structures have confirmed the structural resemblance. [16][17][18] The R39 DD-peptidase is a water-soluble enzyme that is loosely associated with the bacterial cell membrane. Although the precise role of the enzyme in vivo is not known, under in vitro conditions, it has been shown to catalyze the hydrolysis and aminolysis of small D-alanyl peptides and esters.…”

Large ring 1,3-bridged 2-azetidinones: Experimental and theoretical studies

“…Indeed, our reactivity model of serine b-lactamases mimicks the entities involved during the acylation mechanism, namely Ser-70, Lys-73 and Ser-130. These amino acids are also present in the active site of D,D-peptidases, as proven by crystallographic studies of Actinomadura R39 [53] and Streptomyces K15 [54].…”

“…R39 is a model enzyme of low molecular weight D,Dpeptidases [53]. The other considered PBPs are high molecular weight enzymes responsible for bacterial resistance to b-lactam antibiotics:…”