Dictyostelium main page

Links are provided to files on the P450 server.  Some text summarizing the findings is given.  



The ancient model organism Dictyostelium discoideum (cellular slime mold) has 
at least 55 different P450 genes.  See the alignment above.  These include a CYP51
sequence.  CYP524A1 may be the ortholog of CYP61 in fungi.  The other sequences
show no close resemblance to other eukaryotic P450s and they probably evolved 
independently from the CYP51 sequence.  A tree of these sequences (linked above) 
suggests they fall into 15 families.  Assembly of the genes from genomic DNA sequence 
has resulted in assembly of 42 complete P450 genes that probably code for proteins.  There 
are three full length pseudogenes and 10 incomplete pseudogenes.  See the reconciled 
assemblies file above.

Very little is known about the P450 functions in Dicty, however a mutation called RedA in 
the P450 reductase blocks stalk formation.  Stalk formation is dependent on a signaling 
molecule called DIF-1.  This is a hydrophobic chlorinated, hydroxylated compound, a prime 
candidate for a P450 to act on.  Recent work has shown that one and maybe three P450s act 
in the degradation of DIF-1.  DIF-1 is dehalogenated to a molecule called DIF-3 and P450s 
oxidize this molecule.  An abstract of a paper from the Dicty database is shown below.

The proximal pathway of metabolism of the chlorinated signal molecule
DIF-1 in Dictyostelium

Piero Morandini, John Offer, David Traynor, Oliver Nayler, David
Neuhaus, Graham W. Taylor* and Robert R. Kay 

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH,
UK 

*Department of Clinical Pharmacology, Royal Postgraduate Medical
School, London W12 OHS.  

Present addresses.  PM: Dipartimento di Biologia 'L. Gorini", via
Celoria 26, 20133 Milano, Italy.  DT: Department of Pharmacology,
University of Cambridge, Tennis Court Rd, Cambridge CB2 1QJ, UK

Biochemical J., in press.

Abstract

   Stalk cell differentiation during Dictyostelium development is
induced by a chlorinated alkyl phenone called DIF-1.  Inactivation of
DIF-1 is likely to be a key element in the DIF-1 signalling system and
we have shown previously that this is accomplished by a dedicated
metabolic pathway involving up to a dozen unidentified metabolites.
We report here the structure of the first four metabolites produced
from DIF-1, as deduced by mass spectroscopy, NMR and chemical
synthesis.  The structures of these compounds show that the first step
in metabolism is a dechlorination of the phenolic ring, producing DM1.
DM1 is identical with the previously known minor DIF activity, DIF-3.
DIF-3 is then metabolised by three successive oxidations of its
aliphatic side chain: a hydroxylation at w-2 to produce DM2, oxidation
of the hydroxyl to a ketone to produce DM3 and a further hydroxylation
at w-1 to produce DM4, a hydroxy ketone of DIF-3.  We have
investigated the enzymology of DIF-1 metabolism.  It is already known
that the first step, to produce DIF-3, is catalysed by a novel
dechlorinase.  The enzyme activity responsible for the first side
chain oxidation was detected by incubating 3H-DIF-3 with cell-free
extracts and resolving the reaction products by TLC.  It will be
referred to as DIF-3 hydroxylase.  DIF-3 hydroxylase has many of the
properties of a cytochrome P450.  It is membrane bound and uses NADPH
as co-substrate.  It is also inhibited by carbon monoxide, the classic
P450 inhibitor, and by several other P450 inhibitors as well as by
diphenyliodonium chloride, an inhibitor of cytochrome P450 reductase.
DIF-3 hydroxylase is highly specific for DIF-3: other closely related
compounds do not compete for the activity at 100-fold molar excess,
except for a DIF-3 analog lacking the chlorine atom.  The Km for DIF-3
of 47nM is consistent with this enzyme being responsible for DIF-3
metabolism in vivo.  The two further oxidations necessary to produce
DM4 are also performed in vitro by similar enzyme activities.  One of
the inhibitors of DIF-3 hydroxylase, ancymidol (IC50=67nM) will be
particularly suitable for probing the function of DIF metabolism
during development.