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.

Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.

Effect of dietary proline on proline metabolism in the neonatal pig.

The effect of two dietary concentrations of proline (10.3 and 15.8 g/kg) on proline-metabolizing enzymes [pyrroline-5-carboxylate (P5C) reductase and proline oxidase], plasma and tissue free proline concentrations and growth were investigated in the 2- to 13-d-old pig. Diet had no effect on growth or enzyme activity. Diet had a significant (P less than 0.05) effect on the concentration of free proline in plasma, liver, intestine and muscle, but no effect in kidney. These data suggest that the magnitude and pattern of change of P5C reductase activity is not influenced by the concentration of proline in the diet. The lower plasma and tissue free proline concentrations in the piglets fed the basal diet compared with piglets fed the proline-supplemented diet and the lack of effect of diet on enzyme activity suggest there was inadequate proline in the basal diet, and those piglets were unable to increase proline synthesis to maintain normal proline concentrations.

The undifferentiated enzymic composition of human fetal lung and pulmonary tumors.

The concentrations of 16 to 21 enzymes, representing various metabolic pathways, have been determined in human adult, fetal, and neoplastic lung. At midgestation, 12 enzymes (among them, several that metabolize amino acids) were above their adult values while 3 other enzymes were still at low concentrations. These signs of biochemical immaturity are contrasted and compared with those in fetal human liver and rat lung. The enzymic composition of the 11 human pulmonary tumors studied resembled that of the normal fetal lungs closely; the same 12 enzymes were elevated and the same 2 were decreased (compared to nonneoplastic adult lung) in both. The characteristic abnormality in the overall pattern of enzymes, in the concentrations of individual ones, and in the quality of pyrroline-5-carboxylate reductase was clearly evident in both primary and metastatic tumors. The mean concentrations of 10 enzymes in the tumors were significantly different (higher or lower) from those in the control lungs (p less than 0.001 to less than 0.05). The best markers of neoplasticity were thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, hexokinase, and pyrroline-5-carboxylate reductase. The results demonstrate that quantification of a small battery of enzymes, none of them tissue specific, can distinguish adult human lung from its neoplasms.