The crystal structure of phenoxazinone synthase produced by Streptomyces antibioticus and the crystallization and x-ray analysis of the binary toxin produced by Bacillus sphaericus

Phenoxazinone synthase is an oligomeric multi-copper oxidase produced by Streptomyces antibioticus that is responsible for the six-electron oxidative coupling of two molecules of substituted o -aminophenol to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown the enzyme contains one type I (blue) copper center and at least one type II copper center, however, the exact arrangement of copper centers in this multi-copper oxidase is unknown. The purified hexameric form of phenoxazinone synthase was concentrated to 75 mg/mL and used to grow crystals of two different space groups. Crystals of both space groups were grown at 288 K using the sitting-drop vapor-diffusion method. Native data sets extending to a resolution of 3.35 and 2.30 A have been collected for the R32 and P1 space groups, respectively. The trigonal R32 crystals had unit-cell parameters of a = b = 294.2, c = 109.5 A, alpha = beta = 90.0, gamma = 120.0° and the P1 crystals had unit-cell parameters of a = 109.5, b = 163.5, c = 164.4 A, alpha = 117.0, beta = 95.7, gamma = 107.2°. An initial model was obtained using the Bacillus subtilis spore-coat laccase (CotA) as a molecular replacement search model. The molecular replacement model was modified by altering the CotA residues to reflect the sequence of phenoxazinone synthase, building additional residues not present in the search model, and adding the copper atoms to the copper centers. The resulting model shows the expected hexameric arrangement and the presence of an occupied type I copper center.; The Bacillus sphaericus binary toxin is expressed during the early stages of sporulation and is composed of two separately encoded polypeptides, BinA (41.9 kDa) and BinB (51.4 kDa). The binary toxin forms micro crystalline inclusions inside the mother cell that, once ingested, are solubilized in the alkaline pH of the larval gut. Bacillus sphaericus cultures were grown to complete sporulation, harvested and washed. The binary toxin was solubilized with 50 mM NaOH, purified using column chromatography and crystallized. Crystals were grown using the sitting-drop vapor-diffusion method in a glycine buffered solution containing 0.5 M NaCl and 19.5% PEG 4000 as the precipitant. Native data were collected to a resolution of 2.7 A. The binary toxin crystals belong to the triclinic P1 space group with a = 27.92 A, b = 73.21 A, c = 88.71 A, and alpha = 101.39°, beta = 99.11°, gamma = 90.01°. Heavy atom derivative data were collected and used to generate initial multiple isomorphous replacement maps.