Session: Slide Session: Beta-Lactamase: Structure and Function
Sunday, October 26, 2008: 12:00 AM
Room: Independence E (Grand Hyatt)
Background: To understand how CTX-M-15 extended-spectrum β-lactamase has extended the substrate specificity against ceftazidime as compared with CTX-M-3. Methods: CTX-M-15 gene was cloned from an E. coli clinical isolate. PCR with specific primers for CTX-M-15 gene without signal sequence was performed. The structure predicted by a secondary structure predict program revealed that random coils have much more the N-terminal region than C-terminal region in CTX-M-15. Thus, the His-tag was inserted to the C-terminal region. CTX-M-15 gene without signal sequence was expressed by a pET-30 system. CTX-M-15 was purified by His-Bind column and Mono Q column. Crystal of CTX-M-15 was obtained by the batch-crystallization method and hanging-drop method set up by an automatic crystallization machine at 298 K. X-ray diffraction data from CTX-M-15 crystal have been collected to 1.4 Å resolution using synchrotron radiation. Results: The new strategies of cloning and expression system improved the solubility of the expressed CTX-M-15 protein and facilitated the purification of the protein. 10 mg purified protein was obtained. CTX-M-15 was crystallized as a thin plate with a square using a precipitant solution containing 20% (w/v) polyethylene glycerol, 0.1M bis-tris (pH 6.5), and 0.2 M ammonium sulfate. The crystal belonged to the space group P21, with unit-cell parameters a = 44.44 Å, b = 45.6 Å, c = 117.5 Å, β = 90.000°. An Asp240Gly substitution, which converts CTX-M-3 into CTX-M-15, occurred at the end of B3 strand of CTX-M-15. This substitution appeared to increase the mobility of this strand and then led to the improved activity against ceftazidime. Conclusions: These results reveal that the mobility of B3 strand is an important substrate specificity determinant in class A β-lactamases.