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Purification and Characterization of a pyrrole-2-carboxylate Oxygenase From Arthrobacter Strain Py1

Purification and Characterization of a pyrrole-2-carboxylate Oxygenase From Arthrobacter Strain Py1

K Hormann, J R Andreesen

Biol Chem Hoppe Seyler. 1994 Mar;375(3):211-8.

PMID: 8011178

Abstract:

Pyrrole-2-carboxylate oxygenase was purified 8.2-fold to homogeneity from Arthrobacter strain Py1 grown on pyrrole-2-carboxylate as sole carbon, nitrogen, and energy source. FAD and dithioerythritol had to be present during the purification procedure to stabilize the enzyme activity. The molecular mass of the pyrrole-2-carboxylate oxygenase was about 160 kDa by gel filtration chromatography and native gradient PAGE, only one polypeptide of about 60 kDa was present after SDS-PAGE. The FAD content was 2.7 to 3.6 mol FAD per enzyme (160 kDa). The non-covalently bound FAD of the pyrrole-2-carboxylate oxygenase was reduced by NADH and reoxidized by oxygen and pyrrole-2-carboxylate. The enzyme exhibited a narrow substrate specificity. Besides pyrrole-2-carboxylate, only pyrrole, pyrrole-2-aldehyde, and indole-2-carboxylate stimulated the oxygen consumption at a very low rate. The enzyme activity was strongly reduced by different sulfhydryl group inhibitors, but it could be restored by 2-mercaptoethanol or dithiothreitol. The content of pyrrole-2-carboxylate oxygenase was about 6% of the soluble protein as determined by antibodies raised against the enzyme. No cross reacting material was present in other bacteria also able to degrade pyrrole-2-carboxylate. A low amount of the enzyme was present in uninduced cells of Arthrobacter strain Py1, although the enzymatic activity was below the detection limit. The N-terminal amino acid sequence of the enzyme did not contain the consensus sequence GXGXXG found to be present close to the N-terminus of many flavin-dependent monoxygenases sequenced so far.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP1224696723	PY1		1224696-72-3	Price

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