On the Biosynthesis of Bacitracin by a Soluble Enzyme Complex from Bacillus licheniformis

Abstract

1. An enzyme (bacitracin synthetase) which catalyzes synthesis de novo of bacitrain A from the L‐isomers of the constituent amino acids has been purified 11 × from Bacillus licheniformis. The enzyme reaction is dependent on ATP and Mg2+ (or Mn2+). When the l‐isomer of glutamic acid, ornithine, phenylalanine or asparagine was replaced by the corresponding d‐isomer, bacitracin synthesis occurred except in the case of d‐ornithine. 2. The partially purified enzyme was fractionated on a Sephadex G‐200 column into two fractions (peak I and peak II) which were both required for bacitracin synthesis. Peak I catalyzed the 32PPi‐ATP exchange reaction in the presence of the l‐isomers of eight of the ten amino acids in bacibracin. These were isoleuoine, cysteine, leucine, glutamic acid, phenylalanine, histidine, aspartic acid and asparagine. Peak II catalyzed the exchange reaction in the presence of the l‐isomers of the remaining two amino acids lysine and ornithine. Aminoacyl‐tRNA synthetases were absent in both peaks. 3. Peak I and peak II had the ability to bind covalently amino acids when incubated with ATP, Mg2+ and one of the l‐isomers found to catalyze the 32PPi‐ATP exchange reaction. Peak I predominantly bound isoleucine, cysteine, leucine, glutamic acid, phenylalanine, histidine, aspartic acid, asparagine. Peak II predominantly bound lysine and ornithine. The binding of one amino acid did not seem to affect the binding of a second amino acid. The experiments suggest that the covalently bound amino acids are thioester linked. In contrast to what was found with gramicidin S and tyrocidine synthetases no [14C]AMP‐ATP exchange reaction was detected in the presence of the amino acids. 4. The labelled amino acids covalently bound to respectively peak I and peak II were intermediates in bacitracin synthesis. 5. After treatment with alkali and alkaline phosphatase, peak I liberated pantothenic acid which was determined using Lactobacillus plantarum. The results suggest that bacitracin synthetase contains covalent bound phosphopantetheine. Copyright © 1974, Wiley Blackwell. All rights reserved