Journal of Bacteriology, January 2001, p. 109-118, Vol. 183, No. 1
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183-1.109-118.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Department of Microbiology,1 Center for Metalloenzyme Studies,2 and Department of Chemistry,3 University of Georgia, Athens, Georgia 30602
Received 3 July 2000/Accepted 6 October 2000
The two-component anthranilate 1,2-dioxygenase of the bacterium
Acinetobacter sp. strain ADP1 was expressed in
Escherichia coli and purified to homogeneity. This enzyme
converts anthranilate (2-aminobenzoate) to catechol with insertion of
both atoms of O2 and consumption of one NADH. The terminal
oxygenase component formed an
3
3 hexamer
of 54- and 19-kDa subunits. Biochemical analyses demonstrated one
Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center
in each large oxygenase subunit. The reductase component, which
transfers electrons from NADH to the oxygenase component, was found to
contain approximately one flavin adenine dinucleotide and one
ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the
combined components were measured as rates and quantities of NADH
oxidation, substrate disappearance, product appearance, and
O2 consumption. Anthranilate conversion to catechol was
stoichiometrically coupled to NADH oxidation and O2
consumption. The substrate analog benzoate was converted to a
nonaromatic benzoate 1,2-diol with similarly tight coupling. This
latter activity is identical to that of the related benzoate
1,2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously
found to convey temperature sensitivity in vivo because of a
methionine-to-lysine change in the large oxygenase subunit, was
purified and characterized. The purified M43K variant, however, did not
hydroxylate anthranilate or benzoate at either the permissive (23°C)
or nonpermissive (39°C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to
broad-substrate specific dioxygenases that do. Phylogenetic trees of
the
and
subunits of these terminal dioxygenases that act on
natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.
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