title: Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase.
abstract: 2,3-Dihydroxybiphenyl 1,2-dioxygenase, an enzyme of the biphenyl biodegradation pathway that cleaves the first of the aromatic rings, was purified to apparent homogeneity from Pseudomonas sp. strain LB400 that had been engineered to hyperexpress the bphC gene. The enzyme had a subunit molecular mass of 33.2 kDa as determined by SDS-polyacrylamide electrophoresis. Kinetic studies indicate a KM of 7 +/- 1 microM for 2,3-dihydroxybiphenyl. The enzyme is strongly inhibited by substrate (Kss = 300 +/- 10 microM). Catechol, 3-methylcatechol, and 4-methylcatechol were cleaved less efficiently and showed weaker substrate inhibition. 3,4-Dihydroxybiphenyl was not a substrate for the enzyme. Ammonium sulfate and polyethylene glycol 6000 were used as precipitants to obtain two different crystal forms. Crystals grown from ammonium sulfate and polyethylene glycol 6000 had space groups of P4(2)2(1)2 and I222, respectively. Electron microscopy indicates that the enzyme is an octamer (265 kDa) consisting of subunits arranged in two planar tetramers in a staggered conformation.
authors.affiliation: GBF, National Research Center for Biotechnology, Braunschweig, Federal Republic of Germany.
authors.forename: L D
authors.initials: LD
authors.lastname: Eltis
authors.forename: B
authors.initials: B
authors.lastname: Hofmann
authors.forename: H J
authors.initials: HJ
authors.lastname: Hecht
authors.forename: H
authors.initials: H
authors.lastname: Lünsdorf
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000595:Amino Acid Sequence; D003461:Crystallography; D049308:Dioxygenases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase