Phenoxazinone Synthetase from Streptomyces antibioticus : Multiple Activities of the Enzyme

Abstract

A procedure for the preparation of relatively large quantities of highly purified phenoxazinone synthetase from Streptomyces antibioticus is described. Enzyme preparations consisted of multiple forms, as determined by polyacrylamide gel electrophoresis. Each of the electrophoretically separable forms catalyzed the oxidation of catechols, ferrocyanide, and ethylenic thiols, in addition to o -aminophenols.

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

“…Our data suggest that PHS represents l-2% of the total protein present in 48-h 5'. antibioticus cells which is comparable to the values which can be calculated from the data of Katz and Weissbach and Golub and Nishimura (9,11).…”

“…Further, as has already been stated, the purification procedure is easy to scale up or down to meet specific preparative needs. Previously published purification procedures (9,11,23) took several days and generally required far larger quantities of starting material to yield enzyme amounts comparable to those obtained by the procedure reported here. Indeed, Katz and Weissbach obtained only about 3 mg of enzyme/100 g of mycelia ( 9) and Golub and Nishimura obtained only about 4 mg of enzyme/100 g of my-Celia (11).…”

“…Previously published purification procedures (9,11,23) took several days and generally required far larger quantities of starting material to yield enzyme amounts comparable to those obtained by the procedure reported here. Indeed, Katz and Weissbach obtained only about 3 mg of enzyme/100 g of mycelia ( 9) and Golub and Nishimura obtained only about 4 mg of enzyme/100 g of my-Celia (11). Yields as high as 30-50 mg/lOO g of mycelia can be obtained by the procedures described in the present report.…”

“…However, it was not clear from their data that the enzyme preparation they obtained was homogeneous, nor did their purification scheme take into account the possible existence of multiple forms of the enzyme. Indeed, Golub and Nishimura detected the presence of several forms of the enzyme by gel electrophoresis (11). In view of these results, and because of the need for highly purified enzyme for use in the regulatory studies which are ongoing in this laboratory, it seemed necessary to devise a procedure for the complete purification of S. antibioticus phenoxazinone synthetase.…”

Preparation and characterization of a highly stable phenoxazinone synthase nanogel

“…Phenoxazinone synthase (PHS, EC 1.10.3.4) is a bacterial laccase-like multicopper oxidase firstly described by Katz and Weissbach [ 1 ]. As a key enzyme for actinomycin D biosynthesis in Streptomyces , the properties of PHS were preliminarily characterized originally by Golub and Nishimura [ 2 ]. They found it can catalyze oxidation of catechols, ferrocyanide, and ethylenic thiols, in addition to o -aminophenols, which was similar to laccase.…”