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

Abstract

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 reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.

Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source

“…2b). Exogenous addition of 50 mM Fe 2+ to the Ndm enzyme reaction mixture stimulated Ndm enzyme activity, similar to other other Rieske [2Fe-2S]-domain-containing non-haem iron oxygenases such as toluene, naphthalene and biphenyl dioxygenases (Ensley & Gibson, 1983;Subramanian et al, 1979;Yu et al, 2007). Generally, the mononuclear ferrous iron in Rieske oxygenases is not tightly bound and often dissociates during protein purification.…”

“…9816 naphthalene dioxygenase system (Haigler & Gibson, 1990a, b) did not support the N-demethylase reaction by Ndm. In contrast, the reductase components of toluene dioxygenase of P. putida (Subramanian et al, 1979) and biphenyl dioxygenase of Sphingobium yanoikuyae B1 (Yu et al, 2007) are easily substituted by spinach ferredoxin reductase and the reductase of naphthalene dioxygenase, respectively. A proposed reaction scheme for N-demethylation of paraxanthine (and other methylxanthines) by Ccr plus Ndm is presented in Fig.…”

“…However, this remains to be substantiated via cloning of NdmA and NdmB individually. Some Rieske oxygenases are known to be in hexameric a 3 b 3 configuration (Dong et al, 2005;Friemann et al, 2005;Kauppi et al, 1998;Yu et al, 2007) but only the a subunits contain the [2Fe-2S] cluster and the mononuclear ferrous iron, while the b subunits are mainly for structural purposes (Ferraro et al, 2005). The 2-oxo-1,2-dihydroquinoline 8-monooxygenase of P. putida 86 was reported to be in an a 6 configuration, determined by gel filtration chromatography (Rosche et al, 1995), but the crystal structure showed that it is actually a homotrimer (Martins et al, 2005).…”

“…This reductase component oxidized NAD(P)H and concomitantly reduced cytochrome c in vitro; hence, it was designated Ccr (cytochrome c reductase component). The reductases of several well-characterized Rieske non-haem iron oxygenases are also known to reduce cytochrome c in vitro (Subramanian et al, 1979;Haigler & Gibson, 1990b;Yu et al, 2007). Partially purified Ccr was used for the purification of the Ndm component, and the Ccr requirement for N-demethylation by Ndm was specific.…”

“…Ndm is predicted to be a Rieske [2Fe-2S]-domain-containing nonhaem iron oxygenase, and oxygenases of this type are known to be promiscuous in terms of the partner reductases (Subramanian et al, 1979;Yu et al, 2007). However, Ndm did not function in the presence of the reductase and ferredoxin components of Pseudomonas sp.…”