Wilson A. Francisco

Isolation and enrichment of secreted proteins from filamentous fungi.

Filamentous fungi have been recognized as extraordinary producers of secreted proteins and are known to produce novel proteins and enzymes through dispensable metabolic pathways. Here, methods are described for the isolation and enrichment of samples of secreted proteins from cultures of filamentous fungi for analysis by gel electrophoresis and mass spectrometry techniques. These methods can be readily applied to the study of differential protein expression and secretion and metabolic pathways in filamentous fungi by proteomic approaches.

Crystallization and initial X-ray analysis of phenoxazinone synthase from Streptomyces antibioticus

Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown. As a first step towards determining the three-dimensional structure of the enzyme, phenoxazinone synthase has been crystallized. The hexameric form of phenoxazinone synthase was purified from 72 h cultures of S. lividans containing the plasmid pIJ702. Purified hexamers were concentrated to 75 mg ml(-1) and used to grow two forms of crystals. Data collected from the two crystal forms were processed in two separate space groups. Crystals of both forms were grown at 288 K using the sitting-drop vapour-diffusion method. Native data sets extending to resolutions of 3.35 and 2.30 A have been collected and processed in space groups R32 and P1, respectively.