Structure of Phenoxazinone Synthase from Streptomyces antibioticus Reveals a New Type 2 Copper Center^,

Abstract

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.

Direct Identification of a Bacterial Manganese(II) Oxidase, the Multicopper Oxidase MnxG, from Spores of Several Different Marine Bacillus Species

“…One is not redox active and therefore catalytically irrelevant (27), and the function of the other type 1 Cu, although it is redox active, is unclear. Other MCOs with extra Cu atoms include CueO (E. coli), where the extra Cu is involved in regulation (40), and phenoxazinone synthase, where the extra Cu is thought to stabilize quaternary structure (45). These cases are distinguished from MnxG because the Cu atoms are coordinated by histidines and methionines that are not homologous to the consensus MCO Cu-binding regions.…”

Structure and Function of the Engineered Multicopper Oxidase CueO from Escherichia coli—Deletion of the Methionine-Rich Helical Region Covering the Substrate-Binding Site

“…30 The archetypal MCO consists of about 500 amino acid residues and has the three domains, each of which basically has an eight-stranded Greek key β-barrel fold (cupredoxin fold). 31 Cupredoxins are the family of small blue copper (type I Cu) proteins (M r ≈ 14 kDa) characterized by the very intense Cys(S -)→Cu(II) charge transfer transition band in the visible spectrum (λ max ≈ 600 nm, ε ≈ 5,000), high redox potential (+180 ~ +680 mV) and the narrow hyperfine splitting in the EPR spectrum (A Z ≈ 4~8…”

Geometric and electronic structure differences between the type 3 copper sites of the multicopper oxidases and hemocyanin/tyrosinase

“…MCOs use a minimum of four Cu centers: a ''blue,'' type 1 Cu site and a trinuclear Cu cluster composed of a ''normal,'' type 2 Cu and a binuclear type 3 Cu site that together catalyze the 4-electron reduction of O 2 to water with concomitant oxidation of substrates (2,3). Crystal structures indicate that both the type 3 Cu active sites in Hc/CatO/Tyr (4-10) and the MCOs (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) are similarly held in the protein by three His ligands on each Cu center. No additional ligands are present in the deoxy forms, whereas oxygen-derived ligands bridge and exchange couple the two Cu(II)s in the oxidized forms.…”