Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins

Abstract

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin- binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high β-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mM-1s-1). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 A 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 "Hatryoshka dolls." One of these domains is composed of a five-stranded β-sheet with two helices on one side, and the other domain is a double three-stranded β-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 β-lactams. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.