Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2.
A manganese-dependent 3,4-dihydroxyphenylactate 2,3-dioxygenase from Arthrobacter globiformis strain CM-2 (MndD) cloned in Escherichia coli has been purified to homogeneity. Sedimentation equilibrium analysis indicates an alpha 4 homotetrameric holoenzyme structure (4 x 38,861 Da). Steady-state kinetic analysis of MndD with a variety of substrates and inhibitors yields very similar relative rates to the known Fe(II)- and Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenases from Pseudomonas ovalis and Bacillus brevis, respectively. Yet, unlike the Fe(II)-dependent enzyme, MndD retains almost all activity in the presence of H2O2 and CN- and is inactivated by Fe(II). ICP emission analysis confirms the presence of 3.0 +/- 0.2 g-atoms Mn (and only 0.7 +/- 0.2 g-atoms Fe) per tetrameric holoenzyme molecule. Comparison of MndD samples with varying metal content, including an apo and partial-apo enzyme preparation, shows a strong positive correlation between specific activity and Mn content. EPR spectra of MndD as isolated exhibit a nearly isotropic g = 2.0 signal having 6-fold hyperfine splitting (A = 95 G) typical of octahedrally coordinated Mn(II) in a protein. Quantitation of the EPR spin yields 3.4 +/- 0.3 g-atoms of Mn(II) per holoenzyme. When exposed anaerobically to its natural substrate, 3,4-dihydroxyphenylacetate (3,4-DHPA), the EPR spectrum undergoes a dramatic change characterized by the attenuation of the g = 2 signal and the appearance of new signals at g = 1.2, 2.9, 4.3, and 16. The g = 4.3 signal displays 6-fold hyperfine splitting (A = 95 G) that unambiguously assigns it to the Mn(II) center. The appearance of these new signals indicates a large increase in zero-field splitting suggestive of a change in ligand coordination to the Mn(II) center. Similarly perturbed signals are seen in the EPR spectra of MndD complexed with the comparably active substrate analog, D,L-3,4-dihydroxymandelate, or the tight-binding inhibitor, p-nitrocatechol, but not in the complexes with weaker binding substrates and inhibitors. The fact that only strong-binding substrates and inhibitors significantly perturb the Mn(II) EPR signal strongly suggests that the substrate coordinates to the Mn(II) center in the catalytic pathway.
Whiting
A K
AK
Department of Chemistry and Microbiology, University of Minnesota, Minneapolis 55455, USA.
Boldt
Y R
YR
Hendrich
M P
MP
Wackett
L P
LP
Que
L
L
D001173:Arthrobacter / Q000201:enzymology*; D050557:Catechol 2,3-Dioxygenase; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008345:Manganese; D010105:Oxygenases; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; D050557:Catechol 2,3-Dioxygenase