Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/69896
Title: Structural study of the active site of class A β-lactamase : mechanism of the catalytic process and its effect on antibiotic resistance
Authors: Pan, Xuehua
Advisors: Zhao, Yanxiang (ABCT)
Keywords: Beta lactamases
Catalysis
Issue Date: 2015
Publisher: The Hong Kong Polytechnic University
Abstract: Class A ß-lactamases readily hydrolyze and inactivate early generation penicillins and cephalosporins. The catalysis is a two-step process involving acylation that leads to the formation of an acyl-enzyme adduct; and deacylation that hydrolyzes the adduct. Both steps require a general base: two residues, Lys73 and Glu166, have been suggested to fulfill this role. Despite extensive studies not all mechanistic details of this catalytic process are fully understood. Here my thesis reports three studies that aim to provide additional information on the catalytic mechanism of Class A ß-lactamases. The first study aims to illustrate the molecular details of ß-lactam hydrolysis by capturing reaction intermediates with time-resolved X-ray crystallography. Toward this purpose the general base residue Glu166 is replaced by His or Tyr. Such replacements lead to significant slowdown of the enzymatic activity so that in situ catalysis within the crystals can be analyzed by X-ray crystallography. We determined a series of E166Y/H structures in complex with cephaloridine that represent distinct steps of the catalytic process. Collectively these structures offer novel atomic details of the proton transfer pathways that are essential for catalysis. These structures also re-affirm the role of Glu166 as the general base for deacylation. The second study focuses on delineating the mechanism of carbapenem binding and hydrolysis by Class A ß-lactamases. Carbapenems, with distinct chemical structure, show slow acylation kinetics and little deacylation activity toward most Class A ß-lactamases. We found that substituting Glu166 with Ser or other residues significantly enhanced its acylation kinetics by ~100-500 times toward carbapenems. The structures of wild-type and E166S ß-lactamase with or without meropenem reveal that E166S replacement weakens the H-bond network within the active site, causing Asn170 to point away thus to avoid steric hindrance with the bulky 6-a-hydroxyethyl group of meropenem. Furthermore this hydroxyl moiety also adopts a favorable conformation, similar to that seen in Class A carbapenemases. Thus the 6-α-hydroxyethyl side chain of carbapenems is critical for their resistance to Class A ß-lactamases.
The third study targets to identify alternative reactions within the active site of Class A ß-lactamases for potential inhibitor design. We hypothesize that the catalytic Ser70 may undergo alternative reactions if the local environment can be altered. Such reactions, if inducible by small molecules, may lead to new inhibition pathway. Toward this purpose, mutations E166R/K were engineered to perturb the active site. Small molecules that are known to modify the catalytic serine in serine-based enzymes were screened. Indeed our results show that E166R/K mutations alter the Ω-loop conformation, thus enabling phenylmethanesulfonylfluoride (PMSF) to react with Ser70 and convert it to dehydroalanine. Such a modification leads to irreversible inactivation of the ß-lactamase, thus offering a potential new route for inhibitor design. In summary, our studies have provided valuable information on the catalytic mechanism of Class A ß-lactamases. In particular, the novel reaction that converts serine to dehydroalanine not only enrich our knowledge about ß-lactamases for translational studies, but also provide intriguing new possibility of serine-related catalysis or modifications that may affect protein structure and function.
Description: xiv, 154 pages : color illustrations
PolyU Library Call No.: [THS] LG51 .H577P ABCT 2015 Pan
URI: http://hdl.handle.net/10397/69896
Rights: All rights reserved.
Appears in Collections:Thesis

Files in This Item:
File Description SizeFormat 
991021952845203411_link.htmFor PolyU Users167 BHTMLView/Open
991021952845203411_pira.pdfFor All Users (Non-printable)7.12 MBAdobe PDFView/Open
Show full item record
PIRA download icon_1.1View/Download Contents

Page view(s)

14
Last Week
0
Last month
Citations as of Jun 18, 2018

Download(s)

2
Citations as of Jun 18, 2018

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.