Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductaseBPH₋B₁, was a 43 kD monomer containing one mol FAD/mol reductaseBPH₋B₁. The ferredoxin component (bphA3), designated ferredoxinBPH₋B₁, was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenaseBPH₋B₁, was a 217 kD heterotrimer consisting of α and β subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenaseBPH₋B₁ per αβ were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxinBPH₋B₁ and oxygenaseBPH₋B₁ each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductaseBPH₋B₁, ferredoxinBPH₋B₁ and oxygenaseBPH₋B₁ diffracted to 2.5 Å, 2.0 Å and 1.75 Å, respectively. The structures of the three proteins are currently being determined.