Publications

• Purification and characterization of a pyrrole-2-carboxylate oxygenase from Arthrobacter strain Py1.

  Hormann K., Andreesen J.R.

  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 ... >> More

  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. << Less

  Biol. Chem. Hoppe-Seyler 375:211-218(1994) [PubMed] [EuropePMC]

• Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp.

  Becker D., Schrader T., Andreesen J.R.

  Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the ... >> More

  Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated. << Less

  Eur J Biochem 249:739-747(1997) [PubMed] [EuropePMC]