Abstract
Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 microM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.
Highlights
Tuberculosis is the world’s leading cause of death in humans from a single infectious agent [1]
Discovery of a Short Chain Mycocerosic Acid Synthase—The mas-disrupted mutant incorporated labeled propionic acid into C22-C26 mycocerosic acids that are shorter than the C28-C32 mycocerosic acids [9]
To seek the enzyme that catalyzes the synthesis of such short mycocerosic acids, cell-free extracts of wild type and mas-disrupted mutant were subjected to DEAE anion exchange chromatography
Summary
Bacterial Strains and Growth Conditions—TICE BCG vaccine of M. tuberculosis var. bovis BCG was purchased from Organon TeknikaCappel. Short Chain Mycocerosic Acid Synthase from M. bovis BCG used as the cell-free extract This solution (600 mg of protein) was loaded onto a DEAE-cellulose (Whatman DE52) column (1.8 ϫ 30 cm) equilibrated with the previously described 0.1 M potassium phosphate buffer containing 1 mM dithioerythritol, pH 7.0. Following a 60-min incubation at 37 °C the reaction was stopped by the addition of 250 l of 10% NaOH and the mixture was heated in a boiling water bath for 10 min; after acidification, the lipids were extracted with chloroform:methanol (2:1) This lipid material was subjected to thin-layer chromatography, with authentic C18 acid as external standard, on Silica Gel G plates using hexane:diethyl ether:formic acid (35:15:1, v/v) as the solvent. To determine the chain length of the products generated from methylmalonyl-CoA incorporation, the chloroform-extractable material was subjected to TLC as described before and the fatty acids recovered were refluxed with 14% BF3 in methanol for 3 h at 57 °C. The molecular weight of SMAS was calculated from a rectilinear plot of relative mobility versus molecular weight
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