Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad.
A new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 1.6 kb DNA fragment was sequenced and an ORF of 954 bp identified. Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (MpcII) from Alcaligenes eutrophus JMP 222. Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage. The enzyme required Fe2+ for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.
Heiss
Gesche
G
Institut für Mikrobiologie Universität Stuttgart, 70569 Stuttgart, Germany.
Muller
Claudia
C
Institut für Mikrobiologie Universität Stuttgart, 70569 Stuttgart, Germany.
Altenbuchner
Josef
J
Institut für industrielle Genetik Universität Stuttgart, 70569 Stuttgart, Germany.
Stolz
Andreas
A
Institut für Mikrobiologie Universität Stuttgart, 70569 Stuttgart, Germany.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002396:Catechols / Q000378:metabolism; D049308:Dioxygenases; D015723:Gene Library; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D007501:Iron / Q000502:physiology; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002396:Catechols; D009282:Naphthalenesulfonates; D011993:Recombinant Fusion Proteins; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase