Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis.
Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M(r) of 54,200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.
Facey
Sandra J
SJ
Institut für Mikrobiologie, Universität Hohenheim, Garbenstraße 30, D-70593 Stuttgart, Germany.
Groß
Frank
F
Institut für Mikrobiologie, Universität Hohenheim, Garbenstraße 30, D-70593 Stuttgart, Germany.
Vining
Leo C
LC
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
Yang
Keqian
K
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
van Pé
Karl-Heinz
KH
Institut für Biochemie, Technische Universität Dresden, Mommsenstraße 4, D-01062 Dresden, Germany.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002701:Chloramphenicol / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D002701:Chloramphenicol; D010544:Peroxidases; C026195:bromide peroxidase