dc.description.abstract | Streptomyces roseochromogenes, NCIB 10984, hydroxylates exogenous
progesterone to 16a hydroxyprogesterone and thereafter in a second phase
bioconversion to 2ß, 16a-dihydroxyprogesterone. Characterisation of this
reaction was carried out at the whole cell level. The cellular components
responsible for this reaction were also purified to homogeneity. S.
roseochromogenes contains a cytochrome P450 and two electron transfer
proteins, roseoredoxin and roseoredoxin reductase. A reconstituted incubation
containing these purified proteins and the natural electron donor, NADH.
produced identical hydroxyprogesterone metabolites as intact cells.
In sodium periodate (Na104) supported incubations, the initial rate of
progesterone hydroxylation was marginally higher than in the natural
reconstituted system but the product yield was significantly lower. The yield
data showed that the reconstituted natural pathway, supported multiple rounds
of hydroxylation in contrast to a likely single round by a minority of P450s in
the periodate reaction.
When S. roseochromogenes was incubated with exogenous progesterone for
25 h the major metabolite, 16a-hydroxyprogesterone was produced in 3.6 fold
excess to the minor metabolite 2ß, 16a-dihydroxyprogesterone. In a
reconstituted system containing highly purified progesterone 16a-hydroxylase
cytochrome P450, roseoredoxin and roseoredoxin reductase, both metabolites
were produced but in a 10: 1 ratio. When S. roseochromogenes was preincubated
with progesterone and the purified components of the hydroxylase
system assayed as before, the ratio of 16a-hydroxyprogesterone to 2ß, 16adihydroxyprogesterone
produced, decreased to 2.8: 1, virtually identical to the
ratio in whole cell biotransformations. Reconstitution assays containing all
combinations of hydroxylase proteins purified from progesterone preincubated
and control cells, identified roseoredoxin as solely responsible for
the observed changes in in vitro metabolite ratios. The fact that the 2.8: 1 ratio
was also obtained when S. roseochromogenes was exposed to cycloheximide
prior to progesterone pre-incubation; pointed to post translation modification
of roseoredoxin. Separation of two isoforms by 2-D isoelectric focusing
supported this proposition.
A partial 10 amino acid sequence was obtained for both the cytochrome P450
and roseoredoxin for the purpose of probe design for eventual cloning. An
amino acid sequence search revealed this P450 to be unique and unlike any
other known P450 sequence. These two proteins were also successfully
crystallised by hanging drop vapour diffusion trials, giving isomorphous
crystals. These crystals will be used for structure determinations pending
further growth. | en_US |