Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components.
In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.
Hurtubise
Y
Y
INRS-Santé, Institut National de la Recherche Scientifique, Pointe-Claire, Québec, Canada.
Barriault
D
D
Powlowski
J
J
Sylvestre
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001713:Biphenyl Compounds / Q000378:metabolism; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C033223:polyhistidine; D006639:Histidine; D010105:Oxygenases; C074161:biphenyl-2,3-dioxygenase; D005287:Ferredoxin-NADP Reductase