On the right panel dimerisation of two CoaBC trimers is shown with CoaC coloured in teal or grey for different trimers

On the right panel dimerisation of two CoaBC trimers is shown with CoaC coloured in teal or grey for different trimers. (CoaBC (CoaC22 is also observed in some of the protomers, but in an open conformation (Fig.?2b). Open in a separate window Fig. 2 X-ray crystal structure of FMN and CTP-bound MsmCoaBC.a Full aspect of the dodecameric CoaBC with CoaC represented in teal and CoaB in gold. b View of a CoaBC dimer with FMN and CTP shown. Each protomer is coloured differently. The CoaC active site flexible flap is highlighted in blue. c In the left panel, a CoaBC trimer is shown with the CoaC coloured in teal and CoaB in gold. On the right panel dimerisation of two CoaBC trimers is shown with CoaC coloured in teal or grey for different trimers. Each CoaB forms a dimer with protomers L67 from different trimers. Open in a separate window Fig. 3 Detailed view of MsmCoaBC active sites and MsmCoaB dimerisation interface.a View of CoaC active site with FMN bound. The active site sits between two protomers of one trimer (gold and pink) and a third protomer from an adjacent trimer (green). Hydrogen bonds are depicted in yellow and -interactions are in blue. b Superposition of a CoaB crystal structure in green, with full-length CoaBC (teal) showing the active site flaps (brown) of the CoaB and CoaC enzymes. c Detailed view of the CTP binding site. Cartoon and residues belonging to each protomer are coloured differently. Hydrogen bonds and -interactions are coloured as in b. Important waters are represented as red spheres and calcium as a green sphere. Calcium coordination is depicted in purple. d CoaB dimerisation interface. Each protomer is coloured as in c. KIAA1819 The CoaB, which still dimerises and is functional when expressed on its own without the CoaB that could help to explain the different observed oligomerisation patterns (Supplementary Fig.?3). The absence of dimerisation for the and somewhat conserved in the sub-order and many other mycobacteria possess a CoaA (type I PanK) as well as CoaX (type III PanK). However, only the type I PanK seems to be active based on studies in CoaA by binding to the ATP site, with CoA being the strongest regulator29,30. Nevertheless, at physiologically relevant levels L67 of CoA there is only a low level inhibition of CoaA30. It is also known that CoaD, the enzyme that catalyses the fourth step of the pathway, is competitively inhibited by CoA and its product dephospho-CoA31,32. However, nothing was known about the regulation of CoaBC in any organism. We therefore examined the effect of CoA and several of its thioesters (acetyl-CoA, malonyl-CoA and succinyl-CoA) on competitive inhibition, non-competitive inhibition, uncompetitive inhibition. Data are presented as average values of three independent experiments with ?SD. Identification of CoaB inhibitors using high-throughput screening Although the CoA biosynthetic pathway is considered an attractive target for drug discovery, CoA pathway inhibitors displaying potent whole cell activity are rare and the few CoaBC inhibitors that have been reported to date are in majority substrate mimicking13,34. In order to identify structure (PDB: 1U7Z) with the 4-phosphopantothenoyl-CMP (purple) intermediate bound is superimposed. This allosteric site is comprised of a large group of hydrophobic residues (I209, F282?and L304 of protomer A and L203, I292, P299 and I302 of protomer B) many of L67 which L67 form hydrophobic interactions with compound 1b (Fig.?7c). Several -interactions between the compound and the protein are also observed and involve the backbone of D281 and the side chain of F282 of protomer A and R207 of protomer B (Fig.?7d). Hydrogen bond interactions are formed with D281 and F282 of protomer A and R207 of protomer B. Water-mediated interactions.