Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Abstract

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl inComamonas testosteroni B-356 and in Pseudomonassp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-paracarbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze themeta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISPBPH of strain LB400 and the β subunit of ISPBPH of strain B-356 (the αLB400βB-356 chimera) and the αB-356βLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the αB-356βLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the αLB400βB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.

Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It

“…Coincidentally, these features are similar to those reported for strain B-356 BPH dox (17). Unlike these two strains, strain LB400 BPH dox shows a preference for 2,2Ј-dichlorobiphenyl, poorly transforms 3,3Ј-dichlorobiphenyl, and is able to catalyze the metapara hydroxylation of 2,2Ј,5,5Ј-tetrachlorobiphenyl (15,17,22,30).…”

Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation

“…It is believed that b subunit of dioxygenase plays a critical role in combination of a subunit (Hurtubise et al 1998). Structural features of both subunits may influence the dimension and shape of the catalytic site to determine which PAHs can be oxygenated, as well as the orientations of the adjacent reactive carbons towards the active site.…”

“…Structural features of both subunits may influence the dimension and shape of the catalytic site to determine which PAHs can be oxygenated, as well as the orientations of the adjacent reactive carbons towards the active site. Hurtubise et al (1998) demonstrated that both a-and b-subunits of the aryl-hydroxylating dioxygenases influence the enzyme-substrate interaction. Thus, we also examined if any heterogeneity of b subunit existed in the coal-tar-contaminated soil.…”

“…The a-subunit of the terminal dioxygenase is thought to be critical for substrate recognition and is more highly conserved than other components of the dioxygenase complex (Kauppi et al 1998). Hurtubise et al (1998) reported that it is not only the a-subunit but also b-subunit that exert some influence on the enzymesubstrate interaction in conjugation with the a-subunit and thus also critical for enzyme activity. Based on the substrate range of these enzymes, nahAc gene (naphthalene dioxygenase a-subunit) appeared to be specific for the degradation of low-molecular-weight (LMW) PAH (two to three ring PAHs) (Zhou et al 2006), whilst the nidA (naphthalene induced pyrene dioxygenase; a-subunit) gene degraded both LMW and HMW PAHs (Kim et al 2006).…”

Molecular characterization of dioxygenases from polycyclic aromatic hydrocarbon-degrading Mycobacterium spp.

“…The α subunit is the catalytic component and contains two conserved regions: the [Fe 2 –S 2 ] Rieske center and the mononuclear iron binding domain, which are involved in the consecutive electron transfer to the dioxygen molecule [6]. Both α and β subunits are necessary for function and in determining the substrate specificity of the dioxygenase [7]. Most information about metabolic pathways, enzymes, and genes involved in PAH degradation comes from studies on Gram‐negative bacteria [4,5,8,9].…”