RESUMO
The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a heterohexameric (αß)(3) Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356) with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356) and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.
Assuntos
Compostos de Bifenilo/metabolismo , Burkholderiaceae/enzimologia , Dioxigenases/química , Dioxigenases/metabolismo , Bifenilos Policlorados/metabolismo , Burkholderiaceae/química , Burkholderiaceae/genética , Domínio Catalítico , Cristalografia por Raios X , Dioxigenases/genética , Modelos Moleculares , Mutagênese , Filogenia , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
Biphenyl dioxygenase from the psychrotolerant bacterium Pseudomonas sp. strain Cam-1 (BPDO(Cam-1)) was purified and found to have an apparent k(cat) for biphenyl of 1.1 +/- 0.1 s(-1) (mean +/- standard deviation) at 4 degrees C. In contrast, BPDO(LB400) from the mesophile Burkholderia xenovorans LB400 had no detectable activity at this temperature. At 57 degrees C, the half-life of the BPDO(Cam-1) oxygenase was less than half that of the BPDO(LB400) oxygenase. Nevertheless, BPDO(Cam-1) appears to be a typical Pseudomonas pseudoalcaligenes KF707-type dioxygenase.
