A regioselective tryptophan 5-halogenase is involved in pyrroindomycin biosynthesis in Streptomyces rugosporus LL-42D005.
The antibiotic compound pyrroindomycin B contains an indole ring chlorinated in the 5 position. The indole ring is probably derived from tryptophan, and thus primers derived from conserved regions of tryptophan halogenases were used to amplify and clone a DNA fragment that was then used to isolate a tryptophan 5-halogenase gene (pyrH) from a cosmid library of the pyrroindomycin producer Streptomyces rugosporus LL-42D005. A gene disruption mutant in the tryptophan 5-halogenase gene no longer produced pyrroindomycin B, but still produced pyrroindomycin A, the nonhalogenated derivative. The halogenase gene could be overexpressed in Pseudomonas fluorescens BL915 DeltaORF1 and was purified to homogeneity by immobilized metal chelate ion affinity chromatography. Chlorinating and brominating activities with tryptophan as a substrate were detected in cell-free extracts and for the purified enzyme.
Zehner
Susanne
S
Institut für Biochemie, Technische Universität Dresden, 01062 Dresden, Germany.
Kotzsch
Alexander
A
Bister
Bojan
B
Süssmuth
Roderich D
RD
Méndez
Carmen
C
Salas
José A
JA
van Pée
Karl-Heinz
KH
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005800:Genes, Fungal; D005822:Genetic Vectors; D018942:Macrolides / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004271:DNA, Fungal; D018942:Macrolides; C091122:pyrroindomycin B; D010088:Oxidoreductases; C407363:tryptophan halogenase