Purification and characterization of a pyrrole-2-carboxylate oxygenase from Arthrobacter strain Py1.
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.
Hormann
K
K
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Andreesen
J R
JR
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010101:Oxygen Consumption / Q000502:physiology; D010105:Oxygenases / Q000378:metabolism; D011762:Pyrroline Carboxylate Reductases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013379:Substrate Specificity
D005182:Flavin-Adenine Dinucleotide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D011762:Pyrroline Carboxylate Reductases