PI: Nordlund (SU)
Project: Structural studies on protein phosphatases
Funding ends: 31 December, 2000
Structural studies of enzymes in the deoxyribonucleotide
metabolism provide important information for understanding
the structural basis for regulation, specificity and
catalytic mechanism of these enzymes, and is therefore
helpful for the rational design of drugs targeted at
the nucleotide metabolism
Three enzymes in the de deoxyribonucleotide metabolism
have been studied. Most of the efforts of the present
project have been spent in the expression and purification
of these enzymes.
* Ribonucleotide Reductase: A mutant R2 subunit of class
I Ribonucleotide Reductase has been purified and crystallised
and the structure determined using molecular replacement
methods. Refinement of this R2 mutant has been finished
and is now part of a more comprehensive study of iron
free forms of this enzyme. The structure have provide
information which help in understanding the structural
basis for the protonation behaviour of buried carboxylate
groups with implications for proton transfer and metal
binding. A manuscript has been submitted including
this work (see below)
* Deoxyribonucleoside kinase: A purification procedure
for a Drosophila nucleotide kinase has been developed
and the protein purified to homogeneity. Crystallisation
trials have been made but no useful crystals have been
obtained. Another group has recently solved the structure
of the same enzyme.
* 5'-deoxyribnucleotidase: Extensive efforts have been
made into expressing and purifying a cytosolic 5'deoxyribonucleotidase.
A large number of expression vectors have been screened
using the univector recombination cloning system. Recently,
pure protein from a well expressing 5-deoxyribonucleotidase
have been obtained and crystallised. The crystals belong
to a trigonal space group, are well ordered and diffracts
to better than 2.5 Å resolution. Native data
has very recently been collected to 2.7 Å resolutions
and search for useful heavy atom derivatives is in
progress.
Agnes Rinaldo-Matthis has been on maternity leave from August 2000 until January 2001.
Paper
Protonation behaviour of carboxylate residues in the
interior of proteins: Structural and mutagenesis studies
of the carboxylate cluster of ribonucleotide reductase
R2: Xiao-Dong Su, Martin E. Andersson, Agnes Rinaldo-Matthis,Wolfgang
Blodig, Bert-Ove Persson, Britt-Marie Sjöberg
and Pär Nordlund. Submitted to Biochemistry.
Latest update at 5 March, 2001.