Bacilysin biosynthesis by a partially-purified enzyme fraction from Bacillus subtilis

Biosynthesis of dipeptide antibiotic bacilysin by a partially purified enzy me prepared from Bacillus subtilis PY79 was studied. Cell material was desi ntegrated by treatment with lysozyme and sonication and the extract was sub jected to ammonium sulfate fractionation. Bacilysin-synthesizing enzyme act ivity was precipitated between 40% to 70% ammonium sulfate saturation. In v itro enzymatical synthesis of bacilysin was confirmed by performing thin la yer chromatographic comparison of the antibiotic formed with the authentic bacilysin. An enzyme fraction (ca. 125 kDa) was prepared by fast flow gel p ermeation chromatography which was further purified by anion exchange FPLC. The enzymatic synthesis of bacilysin required either ATP or 2'-deoxy ATP a nd was entirely dependent on the presence of constituting amino acids. Alth ough anticapsin, at the concentration used in enzyme assay, did not produce an inhibition zone when assayed against Staphylococcus aureus ATCC 9144, i t exhibited a slight inhibition zone after incubation with the enzyme fract ion in the absence of alanine under the standard assay conditions. To deter mine the mechanism of amino acid activation, ATP-PPi and ATP-P-i exchange r eactions were performed with component amino acids L-alanine and L-anticaps in. The enzyme catalyzed ATP-PPi exchange reaction dependent on L-alanine, but did not activate L-anticapsin in this way. There was also no evidence f or activation of this amino acid as an amino acid phosphate. Pantothenic ac id was liberated from the enzyme fraction as determined microbiologically. Consistently, covalent binding as thioester was shown for L-alanine. These results indicated that the mechanism of bacilysin biosynthesis is not typic al of the general multicarrier thiotemplate model. (C) 2001 Elsevier Scienc e Inc. All rights reserved.