Biosynthesis of bacitracin III. Partial purification of a bacitracin-synthesizing enzyme system from Bacillus licheniformis

Abstract

1. 1. A bacitracin-synthesizing enzyme system was partially purified from the crude extract of Bacillus licheniformis by (NH4)2SO4 fractionation, treatment of calcium phosphate gel, Sephades G-200 gel filtration and hydroxyapatite column chromatography. About a 25-fold purification of the system was achieved. 2. At the step of column chromatography on hydroxyapatite the partially purified system was separated into two distinct components, Peaks I and II, both of which were required for bacitracin synthesis. 3. The enzyme system catalyzed ATP-PPi exchange reactions dependent on the l-isomers of all the bacitracin constituent amino acids. Among the four component d-amino acids, only d-phenulalanine promoted the exchange reaction. 4. The Peak I fraction activated the amino acids constituting the acyclic peptide side-chain of bacitracin and the Peak II fraction activated the amino acids contained in the cyclopeptide moeity. 5. Mixed-substrate experiments suggests that l-isoleucine, l-leucine and l-valine are activated at the same active site(s) as that for l-isoleucine or l-leucine. 6. The activated amino acids seems to be linked as thioesters to the enzyme protein in an intermediate state of bacitracin biosynthesis.