Letno poročilo 2005

Poskusi ekspresije obeh reduktaznih genov so
v teku; oba proteina bomo okarakterizirali v
rekonstituciji z `e omenjeno benzoatno-phidroksilazo
ter nadaljevali z ugotavljanjem
inducibilnosti komponent hidroksilaznega
sistema. S supresivno subtrakcijsko hibridizacijo
smo pridobili odvzemno cDNA
knji`njico in med nukleotidnimi zaporedji 452
klonov odkrili 2 zaporedji, podobni hidroksisteroidni
17beta-dehidrogenazi, ter 12
zapisov za citokrome iz dru`ine P450, od teh
kar 7 podobnih glivnim citokromom z
neznano funkcijo. Nadaljujemo z izolacijo
ustreznih cDNA in funkcijskimi analizami.
Omenjene poskuse spremljajo tudi poskusi
uti{anja genov v glivi C. lunatus, da bi
ugotovili njihovo esencialnost, kot podlago
za konstrukcijo ustreznih inhibitorjev
omenjenega fitopatogenega in oportunega
~love{ko patogenega mikroorganizma.
Nadaljujemo tudi s poskusi direktnega iskanja
samega gena za steroidno 11beta-hidroksilazo.
- V sklopu preu~evanja molekul, ki sodelujejo
pri prenosu signalov s pomo~jo cikli~nih
nukleotidov, nas predvsem zanimajo biokemijske
in strukturne zna~inosti fosfodiesteraz,
ki razgrajujejo cikli~ne nukleotide.
Na tem podro~ju sodelujemo s skupino prof.
dr. Visweswariah z Indian Institute of Science
iz Bangalorja v Indiji. Rekombinantne proteine
iz bakterije M. tuberculosis smo izrazili v E.
coli ter jih izolirali in o~istili v zadostnih
koli~inah za kristalografske in ostale biofizikalne
raziskave. O~i{~ene proteine smo
kristalizirali; dolo~evanje in analiza kristalnih
struktur je v teku.
- [tudije inozitol heksakis-fosfatne kinaze smo
raz{irili z enega encima na vse tri obstoje~e
izooblike. Izdelali in optimizirali smo metodo
za ekspresijo rekombinantnih sesalskih proteinov
v E. coli ter razvili metodo izolacije ~istih
proteinov za nadaljnje biokemijske in biofizikalne
{tudije. Na{e raziskave so bile
usmerjene v podrobne {tudije encimskih
Laboratorij za biosintezo in biotransformacijo
Laboratory for Biosynthesis and Biotransformation
inducibility of different components of the
hydroxylase system are in progress. With suppressive
subtraction hybridization 452 nucleotide
sequences were obtained, among them
2 sequences similar to hydroxysteroid 17beta-dehydrogenase,
and 12 sequences of
the P450 cytochrome family, 7 out of them
similar to fungal cytochromes with unknown
function. We continue with isolation of adequate
cDNA and functional analyses. These
experiments are coupled with gene deletion
experiments in the fungus C. lunatus, to find
out whether these genes are essential, which
could further aid the construction of suitable
inhibitors for this phytopathogen and opportunistic
human pathogenic microorganism.
We continue also with alternative approaches
to discover the gene for steroid 11beta-hydroxylase.
- In the last year we have expanded our studies
of the inositol hexakisphosphate kinase
(IP6K) from one to all three known isoforms
of this enyzme. We established a protocol
for the expression and purification of the
mammalian enzymes in E. coli. The purified
enzymes were used further in the biochemical
and biophysical studies. We extensively
studied the in vitro enzymatic reactions,
analyzed them and compared the product
formation by all three enzymes. Additionally,
we purified the major product of the kinase
reaction and its structure was analyzed by
the nuclear magnetic resonance (NMR). This
is the first structural analysis on the product
of the IP6K reaction so far. This product is a
pyrophosphate form of the fully phosphorylated
inositol ring that is known to be involved
in the phosphorylation of other proteins.
Other physico-chemical features of
these molecules are currently under study.
- Within this project studying the signal transduction
via cyclic nucleotides as the second
messengers, we are particularly interested in
the biochemical and structural features of the
phosphodiesterases, enzymes that degrade
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