title: Expression and homology modeling of 2-aminobiphenyl-2,3-diol-1,2-dioxygenase from Pseudomonas stutzeri carbazole degradation pathway.
abstract: The enzyme 2'-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an alpha(2)beta(2) heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1,082-base pair polymerase chain reaction product corresponding to carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The alpha(2)beta(2)-heterotetrameric 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.
authors: Laboratório de Bioprocessos, Programa de Engenharia Química, COPPE, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. ariane@peq.coppe.ufrj.br
authors: Ariane Leites
authors: AL
authors: Larentis
authors: Rodrigo Volcan
authors: RV
authors: Almeida
authors: Shaila Cíntia
authors: SC
authors: Rössle
authors: Alexander Machado
authors: AM
authors: Cardoso
authors: Welington Inácio
authors: WI
authors: Almeida
authors: Paulo Mascarello
authors: PM
authors: Bisch
authors: Tito Lívio Moitinho
authors: TL
authors: Alves
authors: Orlando Bonifácio
authors: OB
authors: Martins