Purification and characterization of the acyltransferase involved in biosynthesis of the major mycobacterial cell envelope glycolipid – Monoacylated phosphatidylinositol dimannoside

Abstract

Phosphatidylinositol mannosides are essential structural components of the mycobacterial cell envelope. They are implicated in host-pathogen interactions during infection and serve as a basis for biosynthesis of other unique molecules with immunomodulatory properties- mycobacterial lipopolysaccharides lipoarabinomannan and lipomannan. Acyltransferase Rv2611 is involved in one of the initial steps in the assembly of these molecules in Mycobacterium tuberculosis - the attachment of an acyl group to position-6 of the 2-linked mannosyl residue of the phosphatidylinositol mannoside anchor. Although the function of this enzyme was annotated 10 years ago, it has never been completely biochemically characterized due to lack of the pure protein. We have successfully overexpressed and purified MSMEG_2934, the ortholog of Rv2611c from the non-pathogenic model organism M. smegmatis mc2155 using mycobacterial pJAM2 expression system, which allowed confirmation of its in vitro acyltransferase activity, and establishment of its substrate specificity.

Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA

“…A close interaction of the enzyme with the cytosolic face of the mycobacterial plasma membrane might be a strict requirement for PIM 1 or PIM 2 modification by PatA. Supporting this notion, PatA was found to co-localize with the mycobacterial membrane fraction 19 28 . Analysis of the amino-acid sequence of the enzyme revealed the lack of a signal peptide or hydrophobic transmembrane segments, suggesting that PatA associates to only one side of the lipid bilayer, a typical feature of peripheral and monotopic membrane proteins 24 29 .…”

“…Docking calculations placed the polar head of PIM 2 , the better substrate of PatA making important interactions within a region located at the end of the main groove and comprising helices α 4 and α 8 , and the connecting loops β 1 –α 7 (residues 83–90), β 2 –α 8 (residues 148–154) and α 11 –α 12 (residues 282–291; Fig. 6 ) 13 28 . As a consequence, the O6 atom of the Man p ring linked to position 2 of myo -inositol in PIM 2 is predicted to be positioned favourably for activation by H126 and to receive the palmitate group from palmitoyl–CoA ( Fig.…”

“…A truncated version of PatA lacking the first 12 residues of the protein (PatA; MSMEG_2934) 28 , was purified as previously described with the following modifications. M. smegmatis mc 2 155 cells transformed with the corresponding plasmid pJAM2- patA were grown in MM63 medium (15 mM (NH4) 2 SO 4 , 10 mM KH 2 PO 4 , 18 μM FeSO 4 .7H 2 O, pH 7.0) supplemented with 1 mM MgSO 4 , 0.025% (v/v) tyloxapol and 0.2% (w/v) succinate and 20 μg ml −1 of kanamycin.…”

“…PatA transferase activity was measured in the assay with mycobacterial membranes 28 . Briefly, M. smegmatis mc 2 155 cells were broken by sonication and the membrane (100,000 g pellet) fraction was obtained by differential centrifugation.…”

N -Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

“…However, the results were unsatisfactory. Based on our successful purification of mycobacterial acyltransferase PatA (24,25) using the mycobacterial expression system pJAM2, we then attempted solubilization and isolation of recombinant WecA from induced M. smegmatis pJAM2-MSMEG_4947 cells. However, we were again unable to obtain purified WecA protein in sufficient amounts using this approach.…”