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The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.

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The class A PBP1b from Streptococcus pneumoniae is responsible for glycosyltransferase and transpeptidase (TP) reactions, forming the peptidoglycan of the bacterial cell wall. The enzyme has been produced in a stable, soluble form and undergoes time-dependent proteolysis to leave an intact TP domain. Crystals of this TP domain were obtained, diffracting to 2.2 Å resolution, and the structure was solved by using molecular replacement. Analysis of the structure revealed an ''open'' active site, with important conformational differences to the previously determined ''closed'' apoenzyme. The activesite nucleophile, Ser460, is in an orientation that allows for acylation by b-lactams. Consistent with the productive conformation of the conserved active-site catalytic residues, adjacent loops show only minor deviation from those of known acyl-enzyme structures. These findings are discussed in the context of enzyme functionality and the possible conformational sampling of PBP1b between active and inactive states.Keywords: penicillin-binding protein; crystal structure; peptidoglycan; conformational change; transpeptidase The penicillin-binding proteins (PBPs) are the physiological target of the b-lactam class of anti-bacterials, the major weapon against bacterial infection. It is thus imperative that we understand this class of enzymes, and this has been assisted by high-resolution structures of Abbreviations: ASU, asymmetric unit; EDTA, ethylenediaminetetraacetic acid; GT, glycosyltransferase; IPTG, isopropyl thio-b-D-galactoside; MRSA, methicillin-resistant Staphylococcus aureus; PEG MME, polyethylene glycol monomethyl ether; PBP, penicillin binding protein; TP, transpeptidase; RMSD, root mean square deviation.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Highlights d Crystal structure of GIL01 gp7 has been solved d A hybrid approach provides a model for gp7 scaffolding of LexA d gp7 is seen to interact with phylogenetically distinct Staphylococcus aureus LexA d Structural evidence of a phage factor associating with LexA to modulate the SOS response

We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.

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