Chloroperoxidase from Streptomyces lividans: isolation and characterization of the enzyme and the corresponding gene.
For the first time, a halogenating enzyme which is not known to produce halogenated metabolites has been isolated from a bacterial strain. The gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyces lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After cloning of the gene into Streptomyces aureofaciens Tü24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a simple purification procedure using acid precipitation and hydrophobic interaction chromatography was developed. Thus, 54 mg of homogeneous CPO-L could be obtained from 27 g (wet weight) of mycelium. The native enzyme has a molecular weight of 64,000 and consists of two identical subunits. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonheme chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tü24. CPO-L exhibits substrate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is brominated and chlorinated. The functional chloroperoxidase gene was located on a 1.9-kb SalI DNA fragment. DNA sequence analysis revealed an open reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloroperoxidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 was only 42%.
Bantleon
R
R
Institut für Mikrobiologie, Universität Hohenheim, Suttgart, Germany.
Altenbuchner
J
J
van Pée
K H
KH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002711:Chloride Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D007211:Indoles / Q000378:metabolism; D007526:Isoelectric Point; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D007211:Indoles; D008670:Metals; C030374:indole; D002711:Chloride Peroxidase