Purification and characterization of a novel metal-containing nonheme bromoperoxidase from Pseudomonas putida.
A novel bromoperoxidase was purified to homogeneity from the bacterium Pseudomonas putida IF-3 strain, which produces the antibiotic pyrrolnitrin. The enzyme had a molecular mass of 68,000 and was composed of two identical subunits (33,000). It was specific for I- and Br- and inactive toward Cl- and F- in the monochlorodimedone assay system. The optimum pH of the enzyme was around 4.2 and it rapidly lost its activity below 3.5, but it was stable over of range pH of 4 to 11. The purified enzyme was activated several fold by incubation with only cobalt ions, and did not contain an organic prosthetic group such as heme, flavin and cobalamin. Analyses of prosthetic metal compounds in the enzyme using plasma atomic emission spectroscopy (ICP-AES) combined with mass-spectroscopy (MS) and trace metal determination by high performance liquid chromatography (HPLC)-spectrometry, revealed that the enzyme contained 0.35 +/- 0.1 mol of cobalt ions, 1.0 +/- 0.2 mol of nickel ions, 0.8 +/- 0.2 mol of zinc ions and 2.0 +/- 0.2 mol of ferric iron per mol of enzyme, assuming the molecular weight of 68,000. There was no trace of vanadium in the enzyme, unlike in some nonheme haloperoxidases. Thus the bromoperoxidase of P. putida is a novel nonheme metal-containing bromoperoxidase.
Itoh
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N
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Fukui University, Japan.
Morinaga
N
N
Kouzai
T
T
D000595:Amino Acid Sequence; D001965:Bromides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000032:analysis / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005290:Ferric Compounds / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000032:analysis; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000032:analysis
D001965:Bromides; D005290:Ferric Compounds; D007454:Iodides; D003035:Cobalt; D009532:Nickel; D010544:Peroxidases; C026195:bromide peroxidase; D015032:Zinc