Linked Life Data

8555170

Source:http://linkedlifedata.com/resource/pubmed/id/8555170

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1996-2-27
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
160-70
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Manganese(II)-dependent extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis CM-2.
pubmed:affiliation
Department of Chemistry and Microbiology, University of Minnesota, Minneapolis 55455, USA.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S.