Catalytic properties of the 3-chlorocatechol-oxidizing 2, 3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas sp. strain BN6.
The 2,3-dihydroxybiphenyl dioxygenase from Sphingomonas sp. strain BN6 (BphC1-BN6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of different substrates and the effects of various inhibitors on BphC1-BN6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid. Cell extracts containing C23O-mt2 or BphC2-BN6 converted the relevant substrates with an almost constant rate for at least 10 min, whereas BphC1-BN6 was inactivated significantly within the first minutes during the turnover of all substrates tested. Furthermore, BphC1-BN6 was much more sensitive than the other two enzymes to inactivation by the Fe(II) ion-chelating compound o-phenanthroline. The reason for inactivation of BphC1-BN6 appeared to be the loss of the weakly bound ferrous ion, which is the cofactor in the catalytic center. A mutant enzyme of BphC1-BN6 constructed by site-directed mutagenesis showed a higher stability to inactivation by o-phenanthroline and an increased catalytic efficiency for the conversion of 2,3-dihydroxybiphenyl and 3-methylcatechol but was still inactivated during substrate oxidation.
Riegert
U
U
Institut für Mikrobiologie, Universität Stuttgart, 70569 Stuttgart, Germany.
Heiss
G
G
Kuhm
A E
AE
Müller
C
C
Contzen
M
M
Knackmuss
H J
HJ
Stolz
A
A
D020134:Catalytic Domain; D050548:Catechol 1,2-Dioxygenase; D002396:Catechols / Q000378:metabolism*; D049308:Dioxygenases; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002396:Catechols; D004791:Enzyme Inhibitors; C031136:3-methylcatechol; C031137:3-chlorocatechol; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase; C074272:2,3-dihydroxybiphenyl oxygenase