Phenoxazinone synthase from Streptomyces antibioticus: Purification of the large and small enzyme forms

Abstract

Phenoxazinone synthase has been purified approximately 80-fold from 48-h-old cells of Streptomyces antibioticus. The purification procedure involves streptomycin sulfate and ammonium sulfate precipitations, affinity and Bio-Gel chromatography, and glycerol gradient centrifugation. Two forms of the enzyme are purified by these techniques, a large form (L) with a molecular weight of around 900,000 and a small form (S) with an Mr of about 200,000. Electrophoresis of the large and small forms on sodium dodecyl sulfate-polyacrylamide gels reveals the presence of a single polypeptide chain with a molecular weight of around 100,000. Both L and S appear to be distinct molecular forms of the enzyme since rechromatography on Bio-Gel or recentrifugation on glycerol gradients results in retention of the mobility of the form in question. These procedures do not convert L to S or S to L. The purification procedure described has been used for the preparation of phenoxazinone synthase from 12-, 18-, and 48-h cultures of S. antibioticus. These experiments have shown that the relative amounts of L and S which can be isolated depend on the age of the cells used as starting material. As the cultures age, the relative amount of L which is present increases. This result also suggests that L and S are distinct molecular entities. Antibody to a combined L plus S preparation and to L has been raised in rabbits. Anti-L antibody reacts with both L and S and is capable of completely inhibiting phenoxazinone synthase activity in crude extracts of S. antibioticus cells. Thus, the 100,000 Mr subunits of L and S must have some antigenic determinants in common, although they may not be identical in structure. Oxygen consumption during the formation of cinnabarinic acid from 3-hydroxyanthranilic acid was measured with the Clark oxygen electrode. These experiments showed that both L and S catalyze a reaction involving the consumption of 1.5 mol of oxygen per mole of phenoxazinone formed.