Structural Origins of Oxacillinase Specificity in Class D β-Lactamases

Abstract

cSince the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to ␤-lactamase expression. Class D ␤-lactamases are of particular importance due to their presence in multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type ␤-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D ␤-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.

Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii

“…30 Secondly, in both OXA-23 and OXA-24/40 the proline in question is found in a loop that borders the active site and has been implicated in the modulation of both substrate selectivity and catalytic activity for diverse classes of antibiotics. 10, 12, 13, 17 De Luca et al noted the presence of this proline in a sequence motif typically associated with class D carbapenemases. 10 Lastly, the study that identified OXA-160 ( i.e .…”

“…10, 12, 13, 17 The proline, however, resides near the point where the loop reenters the enzyme core as strand β6, and is thus unlikely to make any direct contacts with substrates. This, and the fact that proline’s unique structural properties would be altered upon conversion to serine, suggested to us that the mutation may lead to extensive structural rearrangements of the β5-β6 loop and thus affect substrate binding indirectly.…”

“…We then introduced mutations that are known to diminish or eliminate deacylation of substrates, 33, 34 and have been used successfully in the past to capture acyl-intermediates in class D enzymes. 7, 17 We used site-directed mutagenesis to introduce a V130D substitution into the bla OXA-160 gene and used it to express and purify the variant enzyme. The protein was then crystallized using hanging drop vapor diffusion, and the crystals that were produced were identical in size and shape to OXA-24/40 crystals prepared in the same manner.…”

“…The protein was then crystallized using hanging drop vapor diffusion, and the crystals that were produced were identical in size and shape to OXA-24/40 crystals prepared in the same manner. 7, 17 After soaking these crystals with various ligands and collecting X-ray diffraction data, we were able to determine the structure of OXA-160 V130D with ceftazidime (2.28 Å; PDB 4X55) and aztreonam (2.30 Å; PDB 4X53) bound in the active site (Table 2). Similarly, we introduced a K82D substitution into bla OXA-225 .…”

“…Purified protein (3.0 mg/ml) was combined with well buffer in a ratio of 3:1 (total drop volume 8 µl), and crystals formed within 1–2 days at room temperature. 17 Crystals of OXA-225 K82D were grown starting with 14.0 mg/ml protein using a well buffer composed of 100 mM NaH 2 PO 4 , 100 mM citric acid, 200 mM NaCl, 20% PEG 8000, pH 4.2 with a ratio of 1:1 for protein to well buffer (total drop volume 8 µl). OXA-160 V130D crystals were soaked in either 25 mM ceftazidime (Sigma) or 25 mM aztreonam (Sigma) in well buffer with 5% sucrose added as a cryoprotectant for ~ 60 minutes and flash-cooled in liquid nitrogen.…”