Figure 1.
Mycolic acid synthesis gene cluster in M. tuberculosis H37Rv.
Cluster is highly conserved in all actinomycetes.
Figure 2.
Approximate schematic of proposed mycolic acid synthesis.
Fas I enzyme and FasII system elaborate the α and mero chains for nascent mycolic acids. Carboxylation by AccD4, AccA3 and AccD5 and activation by FadD32 convert these to their respective acyl-S-CoA and acyl-S-AMP forms. The AT (acyltransferase) domain of Pks13 attaches these via a tioester bond to the phosphopentathiene-modified ACP domains, and facilitates transfer to the KS (ketosynthase) domain. Via Claisen-type condensation and reduction, the two chains are joined to form a mature mycolic acid attached via a thioester to Pks13, with loss of CO2. The mature mycolic acid is then hydrolyzed from Pks13 by either the TE1 domain of Pks13, or an external TE. An external TE2 may function to unclog the Pks13 if it is mis-acylated. The liberated mycolic acid may be transferred to a lipid carrier, such as Myc-PL, via an unkown MT (mycolyltranferase); this may facilitate its transfer across the plasma membrane. Eventually it is tranferred by another MT to TMM. TMM is used as a mycolic acid donor for the acceptors of TMM, to form TDM, or AG, to form mAG. Double-boxed enzymes indicate step involves proteins encoded by genes in the mycolic acid synthesis gene cluster with Rv3802c. Question marks represent activities encoded by unknown enzymes and a possible role of Rv3802. Adapted from references [7], [13], [36], [44].
Figure 3.
Clustal W alignment of Rv3802 with its orthologs in mycobacteria demonstrating high conservation.
Cutinase motif is boxed with asterisk over putative catalytic site amino acids.
Figure 4.
Products of Rv3802c and MSMEG_1403 expression in E. coli.
Left: polyacrylamide gel visualized with Sypro Orange™. Right: immunoblot with streptavidin antibody.
Figure 5.
Hydrolysis of various phospholipid substrates by F. solani cutinase (FS), Snake venom PLA (SV), MSMEG_1403 and Rv3802, demonstrating hydrolysis of all except SM.
Thin layer chromatography using 14C-labeled DPPC = dipalmitoyl phosphatidylcholine labeled on both fatty acids (FA), AAPC = phosphatidylcholine with 14C-arachidonic acid on the sn-2, SM = sphingomyelin 14C-labeled on headgroup, PE = phosphatidylethanolamine 14C-labeled on sn-2 FA, PS = phosphatidylserine 14C-labeled on headgroup. Inset is equivalent aqueous phase of phosphatidylserine assay.
Table 1.
Kinetics of Rv3802 and Rv3802 S87ΔE mutant.
Figure 6.
Hydrolysis of PIM purified on a waters column.
Dot over O represents origin. Lane 1 is purified PIM control. Lanes 3 and 4 contain PIM plus Rv3802 and MSMEG_1403, respectively.
Figure 7.
TLC demonstrating thioesterase activity of Rv3802 on 14C-labeled P-CoA and its inhibition by THL, structure above (adapted from DrugBank, http://www.drugbank.ca/drugs/DB01083).
Lanes: 1) 14C-P-CoA alone, 2–8) serially dilutions decreasing the amount of THL from 12.5–0 µM THL with 3.0 µg of Rv3802. Origin is indicated with an O, start substrate indicated with P-CoA, hydrolysis product indicated with FA (fatty acid).
Figure 8.
Structure prediction of Rv3802 using the Phyre server (http://www.sbg.bio.ic.ac.uk/̃phyre/, Bennett-Lovsey et al, 2008).
Model on left predicts a cutinase with 100% precision; modeling on right predicts a thioesterase with 70% precision. Black arrow represents location of catalytic serine 175.