Abstract
Streptomyces viridochromogenes T��494 produces the antibiotic phosphinothricin
tripeptide (PTT). In the postulated biosynthetic pathway, one reaction,
the isomerization of phosphinomethylmalate, resembles the aconitase
reaction of the tricarboxylic acid (TCA) cycle. It was speculated
that this reaction is carried out by the corresponding enzyme of
the primary metabolism (C. J. Thompson and H. Seto, p. 197-222,
in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry
of Antibiotic Production, 1995). However, in addition to the TCA
cycle aconitase gene, a gene encoding an aconitase-like protein
(the phosphinomethylmalate isomerase gene, pmi) was identified in
the PTT biosynthetic gene cluster by Southern hybridization experiments,
using oligonucleotides which were derived from conserved amino acid
sequences of aconitases. The deduced protein revealed high similarity
to aconitases from plants, bacteria, and fungi and to iron regulatory
proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle
aconitase, AcnA, have 52% identity. By gene insertion mutagenesis,
a pmi mutant (Mapra1) was generated. The mutant failed to produce
PTT, indicating the inability of AcnA to carry out the secondary-metabolism
reaction. A His-tagged protein (Hispmi*) was heterologously produced
in Streptomyces lividans. The purified protein showed no standard
aconitase activity with citrate as a substrate, and the corresponding
gene was not able to complement an acnA mutant. This indicates that
Pmi and AcnA are highly specific for their respective enzymatic
reactions.
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