(197f) Deciphering the Late Steps In FR900098 Biosynthesis

Antibiotics containing C-P bonds, phosphonates and
phosphinates, are a potent group of bioactive compounds.  The phosphonic acid FR-900098
is a novel chemotherapeutic agent for the treatment of malaria and has been
shown to be effective in humans and other animals.  This compound inhibits
1-deoxy-D-xylulose 5-phosphate (DXP)
reductoisomerase, the first committed enzyme in the nonmevalonate pathway for
isoprenoid biosynthesis in many bacteria as well as Plasmodium falciparum,
the parasite responsible for the most virulent form of the disease. The biosynthetic
cluster for FR-900098 has recently been cloned from Streptomyces
rubellomurinus and heterologously expressed in Streptomyces lividans. 
The gene cluster was also successfully reconstituted in E. coli, resulting
in 6 mg/mL FR-900098.

The initial step in FR-900098 biosynthesis is similar to
almost all other known phosphonate biosyntheses, a phosphoenolpyruvate (PEP)
mutase catalyzed reaction of PEP to phosphonopyruvate.  The subsequent steps
parallel the tricarboxylic (TCA) cycle, resulting in the formation of
2-oxo-4-phosphonobutyrate, an analog of α-ketoglutarate.  The final steps
were proposed based only on sequence homology of the remaining genes, without
any definitive assignment of the order or function.  Here we report the deciphering
of the last steps in FR-900098 biosynthesis, verified through whole cell
feeding experiments and in vitro assays with purified enzymes.  Two
novel enzymes were revealed in the process; one a bifunctional enzyme capable of
performing a nucleotide transfer and decarboxylation and the other an amide
hydroxylase.  A complete understanding of the FR-900098 biosynthetic pathway
now opens the possibilities for metabolic engineering in the native or
heterologous Streptomyces strain or in E. coli to increase
production of the antibiotic and combinatorial biosynthesis to generate novel
derivatives of FR-900098.