Abstract
Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide. The class A serine β-lactamase BlaC confers Mycobacterium tuberculosis resistance to conventional β-lactam antibiotics. As the primary mechanism of bacterial resistance to β-lactam antibiotics, the expression of a β-lactamase by Mycobacterium tuberculosis results in hydrolysis of the β-lactam ring and deactivation of these antibiotics. In this study, we conducted protein X-ray crystallographic analysis of the inactivation of BlaC, upon exposure to the inhibitor bis(benzoyl) phosphate. Crystal structure data confirms that serine β-lactamase is phosphorylated at the catalytic serine residue (Ser-70) by this phosphate-based inactivator. This new crystallographic evidence suggests a mechanism for phosphorylation of BlaC inhibition by bis(benzoyl) phosphate over acylation. Additionally, we confirmed that bis(benzoyl) phosphate inactivated BlaC in a time-dependent manner.
Highlights
According to the 2017 World Health Organization’s (WHO) report, Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide [1]
BlaC, a hydrolase (EC 3.5.2.6), cleaves lactam carbon–nitrogen amide bonds rapidly, effectively deactivating β-lactam antibiotics [22,23]. This β-lactam hydrolysis proceeds through an acylation–deacylation reaction
Lys-73 acts as a general-base catalyzing the nucleophilic Ser-70 attack of the lactam carbonyl carbon and formation of a tetrahedral intermediate [22,23,24]
Summary
According to the 2017 World Health Organization’s (WHO) report, Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide [1]. In the case of the phosphoserine crystal, Ser-70 showed clear density for a phosphoserine having been phosphorylated by the inhibitor bis(benzoyl) phosphate (Figure 3A). The phosphoserine BlaC structure contained phosphate molecules near the active site adjacent to the nucleophilic Ser-70 (Figure 3B). Even though the previously published BlaC structures contain phosphate molecules near the active site, they do not have a phosphorylated Ser-70. As previously reported for BlaC and other class A serine β-lactamases such as TEM-1, SHV-1, and the CTXMs, the global fold consists of two domains, the α-domain and the α/β-domain (Figure 4) [8,18–. The active site exhibits a high degree of conservation for the catalytic residues Ser-70, Lys-73 and Glu-166. The protperionteninamnaemaenadnrderperperseesnentatatitviveePPDDBB IIDD nnuummbbeersrsaraerelisltiesdte. dA.bAovbeotvhee tsheqeuseenqcuee, tnhceep, othsietiponosoiftitohne of the helihceeslic(e~s) (a~n) danbdetbaetsatrsatrnadnsd(s→(→) )araereininddicicaatteedd aanndd nnuummbbeerreedd. .TThehenunculecolepohpilhicilSicerS-7e0r-i7s0hiisghhliigghhltiegdhted in greeinn garnedenthanedcathtaelcyattiaclyLtyics-L7y3s-a7n3danGdluG-l1u6-616a6raerheihgihglhilgighhteteddiinn rreedd
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