Mycobacterial rhomboids are active rhomboid-serine-proteases Multiple sequence alignment revealed that all mycobacteria rhomboids contain the putative rhomboid catalytic residues Gly199, Ser201 and His254. The mycobacterial rhomboids also contained two additional C-terminal Histidins (His145 and His150, which together with His254 are universally conserved in the rhomboid proteins [19]) and five invariant transmembrane residues (Gly202, Linsitinib supplier Gly257, Gly261, Asn154 and Ala200) that are also conserved in many rhomboid proteins [33]. However in mycobacteria, Ala252 which occurs in many eukaryotic

and prokaryotic rhomboids was substituted by Gly (figure 4). Furthermore, Tyr205 which stabilizes the rhomboid protease active site of glpG [17, 33] and of many rhomboid proteases was only conserved in MAB_0026 of M. abscessus, being substituted by Phe in mycobacterial rhomboids (figure 4). Thus, Phe is the stabilizing IWR1 residue in the protease active site for majority of mycobacterial rhomboids (Phe is an additional stabilizing residue for rhomboid proteases [17]). Figure 4 Mycobacterial

rhomboids are active rhomboid proteases. Highlighted in blue are the rhomboid catalytic dyad residues (Ser201 and His254); yellow, the invariant residues in this alignment; grey, the rhomboid family invariant residues that were not conserved in this alignment. Locus tags for mycobaterial rhomboids are boxed blue. Included: aarA [GenBank: L28755] Sclareol of P. stuartii; glpG [GenBank: AAA23890] of E. coli; rho-1 [GenBank: AAF47496.1] of Drosophila; (Ano1) AgaP_AGAP004737 [GenBank: XP_318085] of Anopheles gambiensi; (Tox1) [GenBank: #Q695U0] of Toxoplasma gondii; (Fal1) PF11_0149

[GenBank: XP_001347820] of P. falciparum and RHBDL2 [GenBank: NP_060291.2] of human. The nature of the transmembrane helices (TMHs) formed by mycobacterial rhomboids was analyzed to determine whether they conform to those of active rhomboid proteases. Mycobacterial orthologs of Rv0110 formed seven TMHs and topologies similar to those of eukaryotic rhomboid rho-1 of Drosophila (see figure 5). As in rho-1, the rhomboid catalytic residues GxSx & H (Gly199, Ser201 and His254, × being any residue) were localized respectively, in TMH4 and TMH6 (see figure 5 and details in additional file 1). In mycobacterial orthologs of Rv0110, the two C-terminal histidine and asparagine (His145, His150 and Asn154) were localized in TMH2, in contrast to eukaryotic rhomboid proteases which have these residues in TMH3 [17, 19, 23]. However, in our analyses, we found His145, His150 and Asn154 in TMH2 in rho-1, similar to Rv0110 (see additional file 2).