C1-886. X-Ray Crystallographic Analysis of the Ceftazidime-Hydrolyzing CTX-M-15, Class A Extended-Spectrum β-Lactamase from an Escherichia coli Clinical Isolate
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.
Ha Jung1, Hyun Lee1, Jae Lee1, Jung Lee, MS2, Sang Lee, Dr3, Seung Sohn1 and  S. H. Lee, None., (1)Myongji University, (2)Asan Medical Center, Seoul, Korea, Republic of, (3)Myongji University, Yongin, Korea, Republic of

Current Licenses and Certification
Korean License of Medical Doctor
Education
Mar, 1996 - Feb, 2002
University of Ulsan, College of Medicine, Seoul, Korea
Professional Experiences
Internship
Mar, 2002- Feb, 2003
Asan Medical Center, Seoul, Korea
Residency
Mar, 2004-
Department of Internal Medicine,
Asan Medical Center, Seoul, Korea