Abstract
Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its “resting state” and DPA-bound “active state”. EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is, worldwide, the leading cause of human fatalities due to any infectious disease (WHO, 2018)
Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis
The related NV13 acceptor, where the 3-OH position of the terminal arabinose unit of NV6 is blocked by an azide group was used in subsequent cell-free experiments for purified EmbB, EmbC, and the abundant AftB activity from Mycobacterium smegmatis (Msm) membranes (Lee et al, 1997), to determine the resulting new glycosidic linkage in NV12 catalyzed by EmbB, which is presumably an α(1→3)linkage based on previous studies (Escuyer et al, 2001)
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is, worldwide, the leading cause of human fatalities due to any infectious disease (WHO, 2018). Ethambutol (EMB) is one of the five front-line drugs used to treat TB and is important in MDR-chemotherapy regime (Alliance, 2008). It exhibits its mode of action by inhibiting the biosynthesis of arabinogalactan (AG). (Takayama and Kilburn, 1989; Mikusova et al, 1995; Lee et al, 2005), a key component of the Mtb cell wall mycolylarabinogalactan-peptidoglycan (mAGP) complex (Jankute et al, 2015). Functional studies have shown that all the Emb proteins play key roles in cell wall synthesis. EmbA knockout Msm (斜体) strain can survive, indicating EmbB protein can work alone in cell (Escuyer et al, 2001). We show that ethambutol inhibits the enzymatic activity of the EmbB2-AcpM2 complex and structurally identify the site that is most susceptible to ethambutol resistance based on isolates from clinical studies
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