Purification and characterization of a novel bacterial non-heme chloroperoxidase from Pseudomonas pyrrocinia.
The first bacterial chloroperoxidase that is capable of catalyzing the chlorination of indole to 7-chloroindole was detected in Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin (Wiesner, W., van Pée, K.H., and Lingens, F. (1986) FEBS Lett. 209, 321-324). Here we describe the purification and characterization of this bacterial non-heme chloroperoxidase. The enzyme was purified by DEAE-cellulose chromatography at different pH values, molecular sieve chromatography, and Bio-Gel HTP hydroxylapatite. After the last purification step, chloroperoxidase was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. Based on gel filtration and ultracentrifugation results, the molecular weight of the enzyme was 64,000 +/- 3,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band with the mobility of a 32,000 molecular weight species. Therefore, in solution at neutral pH, this chloroperoxidase is a dimer. The enzyme did not exhibit any absorbance in the visible region of the spectrum. The isoelectric point was 4.1. Chloroperoxidase was specific for I-, Br-, and Cl- and was not inhibited by azide, but was inhibited by cyanide and F-. This procaryotic chloroperoxidase catalyzed the bromination of monochlorodimedone but not its chlorination and has no peroxidase or catalase activity. The pH optimum of the enzyme was between 4.0 and 4.5, and the enzyme was stable between pH 3.5 and 8.5 and showed no loss of activity when incubated at 60 degrees C for 2 h. Chloroperoxidase also chlorinated 4-(2-amino-3-chlorophenyl) pyrrole to yield aminopyrrolnitrin, the immediate precursor of pyrrolnitrin. This suggests very strongly that chloroperoxidase is involved in the biosynthesis of the antibiotic pyrrolnitrin.
Wiesner
W
W
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Federal Republic of Germany.
van Pée
K H
KH
Lingens
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001965:Bromides / Q000378:metabolism; D002711:Chloride Peroxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D002845:Chromatography; D004355:Drug Stability; D005459:Fluorides / Q000494:pharmacology; D007454:Iodides / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification*; D011549:Pseudomonas / Q000201:enzymology*; D011758:Pyrroles / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D001965:Bromides; D002712:Chlorides; D007454:Iodides; D046911:Macromolecular Substances; D008670:Metals; D011758:Pyrroles; C057130:4-(2-amino-3-chlorophenyl)pyrrole; D010544:Peroxidases; D002711:Chloride Peroxidase; D005459:Fluorides