Structural and mechanistic insights into NDM-1 catalysed [beta]-lactam antibiotics via cadmium substitution

Abstract

Bacterial infection is a kind of harmful strain of bacteria on or inside the human body. Illnesses induced by bacterial infections can range from minor diseases like strep throat to life-threatening situations such as encephalitis. -lactam antibiotics represent a class of antibiotics consisting a -lactam ring in their molecule structures and are the mainstay to treat most bacterial diseases. However, -lactam antibiotic resistance mediating by metallo--lactamases (MBL) are starting to emerge. NDM-1, as the abbreviation of New Delhi metallo--lactamase-1, is a newly added member of the MBL family which is able to hydrolyse nearly all -lactam antibiotics and has speedily spread to around the world as well as raised clinical concerns. It is highly desired to find mechanism based as well as broad-spectrum inhibitors against all the MBLs family members. However, various hydrolysis mechanisms of MBLs regarding to different types of antibiotics pose a huge challenge to design inhibitors of MBLs. As the hydrolysis process of -lactam antibiotics by NDM-1 is fast, it is not easy to capture intermediates of substrates. Few previous studies give a clearly mechanistic insight into NDM-1 catalysed -lactam antibiotics based on crystal structures. Thus, to promote the design of mechanism-based inhibitors, in this project, I focus on the crystal structures solving of hydrolysed -lactam antibiotics and related intermediates catalysed by NDM-1, aiming to elucidate the mechanism of hydrolysis process of -lactam antibiotics by NDM-1. Experimental method of cadmium substitution of NDM-1 is applied for slowing down hydrolysis process on the purpose of capturing more states of substrates during hydrolysis. In this project, enzymatic study of NDM-1 and NDM-1-Cd whose zinc ions in the catalytic pocket were replaced by cadmium ions was performed to support that cadmium substitution could reduce enzyme activity of NDM-1. Crystal structure of NDM-1-Cd was solved at 1.25-Å resolution, indicating that cadmium substitution was nearly complete in the active site of NDM-1-Cd and di-metal distance of catalytic pocket becomes longer via structural analysis. What’s more, crystal structure of NDM-1-Cd in complex with mixture of hydrolysed and un-hydrolysed ampicillin belonging to penicillin family was solve at 1.39-Å resolution. Different with previous data reported, most part of the mixture was un-hydrolysed ampicillin. The intermediate obtained is closed to initial state of reaction pathway and the catalytic pocket becomes larger along with reaction proceeding, contributing to propose catalytic mechanism of penicillin by NDM-1. The X-ray crystal structure of NDM-1 in complex with hydrolysed cefuroxime belonging to the family of cephalosporins was solved at1.75-Å resolution. Through analysing the complex, it was demonstrated that water molecule/hydroxide in the active site which is essential for provide an ideal proton donor for anionic intermediates was absence, contributing to propose catalytic mechanism of cephalosporins by NDM-1. In conclusion, cadmium substitution is a helpful method to inhibit enzymatic activity of NDM-1 and can be applied to mechanism study of NDM-1. Moreover, the catalytic mechanism of penicillin and cephalosporins is clearer than previous reports.