|Title:||Generation and kinetic characterization of hybrid beta-lactamases|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||iii, 288 leaves : ill. (some col.) ; 30 cm.|
|Abstract:||The β-lactam antibiotics have been in use for human and animal therapy for more than 50 years. The synthesis of β-lactamases represents the most widespread and the most efficient mechanism devised by the pathogenic bacteria to escape the lethal action of β-lactam antibiotics. Of all β-lactamases, class A enzymes are the most commonly encountered by clinicians and hence, have been extensively studied. However, despite numerous kinetic, structural and site-directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β-lactamases and their surprising catalytic power. The solutions to these problems may represent the cornerstones on which better antibiotics and inhibitors of β-lactamases can be designed, hopefully on a rational basis. The generation and analysis of hybrid enzymes is one of the most powerful tools for understanding structure-function relationships of enzymes and the creation of enzymes with novel properties. Eighteen chimeric β-lactamase genes (six frompenPC andpenP, 12 from penPC and pel) were successfully generated in E. coli RRl by in vivo intramolecular recombination within the homologous regions between two class A β-lactamase genes [the β-lactamase I gene from Bacillus cereits (penPC) and the β-lactamase gene from Bacillus licheniformis (penP); or penPC and the β-lactamase gene from Staphylococcus aureus (pel)]. These hybrid genes encode novel β-lactamases with their N-terminal moiety derived from PenPC and C-terminal moiety derived from either PenP or PCI. While PenPC, PenP and their hybrid β-lactamases were highly expressed in Bacillus siibtilis by the use of a novel expression vector, ø105 MU331 prophage, the efforts to express PCI and its hybrid β-lactamases with the same system were proved to be unsuccessful. The expressed PenPC, HybridB, HybridC, HybridD, HybridE and HybridF were purified to high purity by affinity binding to Celite, but PenP and HybridA were purified by cation-exchange chromatography. PCI and its hybrid β-lactamases were successfully expressed in E. coli as GST fusion proteins. The expressed GST fusion proteins with PCI, PenPC, Hybrid 1, Hybrid2, Hybrids, and Hybrid4 were highly soluble. However, the expressed GST-Hybrid5 was only partially soluble, and the other GST fusion proteins were found to be expressed as inclusion bodies. Many efforts made to improve the solubility of these fusion proteins were demonstrated to be unsuccessful, including lowering the growth temperature during induction from 37 ℃ to 20℃, 25℃ or 30℃, decreasing IPTG concentration to 0.05 mM, expressing fusion protein in different host stains [BL21, BL21(DE3)] and the addition of ethanol (to a final concentration of 3%) to the growth medium. Nevertheless, when all these hybrids were expressed as maltose binding protein (MBP) fusions in E. coli, all the expressed fusion proteins were found to be highly soluble. They were purified by a one-step amylose affinity column. Detailed kinetic characterizations and stability studies were carried out for these purified β-lactamases. Many conclusions are drawn: (1) The C-terminus of PenPC is essential to its stability and effective catalysis. The replacement of it (42 amino acids) with the corresponding sequence from PCI causes significant loss of enzyme activity and stability. The identity and similarity of this C-terminal region between PenPC and PCI are 30% and 62%, respectively, which are quite similar to the identity (34%) and similarity (57%) between the two entire polypeptides; (2) The region in between the crossover points in Hybrids and Hybrid6 may be very important for the determination of substrate specificity of Class A β-lactamases, since the Km values of MBB-Hybid5 (similar to PCI) for many β-lactams are significantly different from those of MBB-Hybrid6 (similar to PenPC). By alignment of protein sequences, the two hybrids differ at a 43-amino-acid region, covering Ser 70 and Lys 73, which are crucial active site residues for Class A β-lactamases. The identity and similarity of this region between PenPC and PCI are 41% and 69%, respectively, which are much higher than those of the two entire polypeptides. Some residues in this region may play important role in determining substrate specificity; (3) Compared with their parents, hybrid enzymes lose their stability to different extent; (4) Some hybrids, especially those with crossovers occurred near the middle of the two β-lactamases, such as Hybrid6, Hybrid7 and Hybrids, dramatically lost their catalytic efficiency in hydrolyzing penicillin G, ampicillin and nitrocefin. In contrary, they have high substrate affinity to other β-lactams with bulky side-chains such as methicillin and oxacillin. This suggests that the generation of hybrid enzymes is similar to the evolution process. In carving out a larger active site to accommodate the β-lactams with bulky side-chains, these new enzymes lost their power to hydrolyze β-lactams with simple side-chains. Also, they lost their internal actions that formerly contributed to their internal integrity, lowering their stability.|
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