title: An insight into the biophysical characterization of different states of cefotaxime hydrolyzing β-lactamase 15 (CTX-M-15).
abstract: Cefotaxime hydrolyzing β-lactamase-15 (CTX-M-15) is encoded by blaCTX-M-15 gene present on plasmid of various Gram-negative bacteria, such as E. coli, E. cloacae, K. pneumoniae, etc. The widespread dissemination of CTX-M-15 harboring bacteria in hospital as well as community settings is a universal threat as they are resistant to various clinically significant antibiotics. In order to gain an insight into the folding mechanism of CTX-M-15, we carried out pH-induced denaturation study by monitoring Trp fluorescence, far-UV circular dichroism (CD), and ANS fluorescence. We found that the pH-induced denaturation of CTX-M-15 was a three-step process with the accumulation of two stable folding intermediates (XI at pH 2.5 and XII at pH 1.5) in the folding pathway. The intermediates were further characterized by far-UV and near-UV CD analysis, Trp fluorescence, ANS fluorescence, three-dimensional fluorescence, acrylamide quenching, dynamic light scattering, and thermal denaturation studies. We found that XI state lacked tertiary structure but retained most of the secondary structure, its Trp residues were partially exposed to the solvent and its hydrophobic patches were highly accessible to ANS. On the other hand, a complete disruption of tertiary structure along with more than 50% loss in secondary structure was observed in XII state. We conclude that the XI state of CTX-M-15 at pH 2.5 had all the characteristics of a molten globule (MG) state, while its XII state at pH 1.5 was more similar to pre-molten globule (PMG) state. ANS fluorescence also showed that the binding of ANS in XII state was lower than that in the XI state. We propose that the accumulation of MG- and PMG-states was due to separation (at pH 2.5) and then unfolding (at pH 1.5) of the αβα-fold of CTX-M-15, respectively.
authors.affiliation: a Medical Microbiology and Molecular Biology Laboratory, Interdisciplinary Biotechnology Unit , Aligarh Muslim University , Aligarh , UP 202002 , India.
authors.forename: Md Tabish
authors.initials: MT
authors.lastname: Rehman
authors.forename: Mohd
authors.initials: M
authors.lastname: Faheem
authors.forename: Asad U
authors.initials: AU
authors.lastname: Khan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002439:Cefotaxime; D016972:Enterobacter cloacae / Q000201:enzymology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C473109:beta-lactamase CTX-M-15; D001618:beta-Lactamases; D002439:Cefotaxime
keywords: extended spectrum β-lactamase; non-native intermediates; protein folding; protein stability; three-dimensional fluorescence