Title: The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation Elamin AA, Stehr M, Spallek R, Rohde M, Singh M Ref: Molecular Microbiology, 81:1577, 2011 : PubMed
Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 microM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.
Title: Purification and characterization of a novel mycolic acid exchange enzyme from Mycobacterium smegmatis Sathyamoorthy N, Takayama K Ref: Journal of Biological Chemistry, 262:13417, 1987 : PubMed
We have isolated and purified to homogeneity an alpha,alpha'-trehalose 6-monomycolate:alpha,alpha'-trehalose mycolyltransferase (trehalose mycolyltransferase) from Mycobacterium smegmatis that catalyzes the exchange of a mycolyl group between trehalose, trehalose 6-monomycolate (TM), and trehalose 6,6'-dimycolate (TD). This enzyme was prominent in M. smegmatis and it catalyzed the following reactions. TM + [14C]trehalose in equilibrium [14C]TM + trehalose [14C]TM + TM in equilibrium [14C]TD + trehalose This enzyme was purified by (i) ammonium sulfate fractionation, (ii) QAE-Sephadex A-50 column chromatography, (iii) gel filtration on a Sephadex G-75 column, and (iv) SP-Sephadex C-50 column chromatography. The purified protein yielded a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 25,000. This enzyme was a glycoprotein, had no cofactor requirement, and was highly specific for alpha,alpha'-trehalose as the mycolate acceptor. It was less specific for the acyl donor group since the palmitoyl group in trehalose 6-monopalmitate was easily exchangeable. There was no TM acylhydrolase activity in the purified enzyme, suggesting that it is probably associated with the anabolic pathway of mycolic acid metabolism. We postulate the formation of a mycolyl-enzyme intermediate in this reaction. Such an intermediate could play a central role in the transfer of mycolic acid to form the prominent cell wall components of mycobacterial TD and possibly murein-arabinogalactan-mycolate.
