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Playing with molecules: in
our studies the key players are the protein molecules and
their ligands. The protein molecules can be very big and
oligomeric or small and monomeric. By mutating the protein
molecules by molecular biology and synthesizing
substrate/ligand analogues by organic chemistry we make a
range of molecules that are being used to probe the
properties of the protein-ligand/substrate interactions of
our studied enzymes. In this way we contribute to a better
understanding of the properties of these fascinating
biological molecules.
PDB-entry: 2X58
The crystal structure of the rat peroxisomal full length
MFE1 shows the assembly of the N-terminal crotonase part with
respect to the C-terminal HAD-part. In the crotonase active
site the ligand CoA is bound, adopting a similar conformation
as seen in the enoyl-CoA hydratase active site. The active
sites of the crotonase and the HAD parts, separated by a
distance of 40Å, are interconnected via a tunnel in which the
positively charged side chains of lysine and arginines are in
excess over the acidic residues, suggesting that positive
charges could facilitate substrate tunneling between the two
active sites.
Kasaragod, P., Venkatesan, R., Kiema. T.R.,
Hiltunen, J.K., Wierenga, R.K. (2010) The crystal structure
of liganded rat peroxisomal multifunctional enzyme type 1: a
flexible molecule with two interconnected active sites. J
Biol Chem, 285, 24089-24098.
The mode of binding of the potassium and chloride ions to
human tetrameric T2-thiolase has been characterized. The
potassium ion is bound near the active site, which explains
why this T2 thiolase is activated by potassium ions. The mode
of binding of the 2-methyl-acetoacetyl moiety of the
substrate in the T2 active site cavity is predicted.
Haapalainen, A.M., Meriläinen, G., Pirilä,
P.L., Kondo, N, Fukao, T., Wierenga, R.K. (2007)
Crystallographic and kinetic studies of human mitochondrial
acetoacetyl-CoA thiolase (T2): the importance of potassium
and chloride ions for its structure and function.
Biochemistry 46: 4305-4321.
PDB-entry: 3GZE
The proline rich peptide substrate (SP)10 is bound to a
monomeric algal prolyl 4-hydroxylase in an extended
polyproline-typeII-like conformation, covered by two loops.
The structure shows how the reactive proline of the substrate
peptide is bound near the catalytic metal ion.
Koski, M.K., Hieta R., Hirsilä, M., Rönkä,
H., Myllyharju, J., Wierenga, R.K., (2009) The crystal
structure of an algal prolyl 4-hydroxylase complexed with a
proline-rich peptide reveals a novel buried tripeptide
binding motif. J Biol Chem 284:25290-25301.
A new monomeric TIM variant (A-TIM) has been obtained with
completely novel binding properties, like being able to bind
citrate in its active site. Nevertheless the wild type
suicide inhibitor, bromohydroxyacetone phosphate, also binds
in the active site.
Alahuhta, M., Salin, M., Casteleijn, M.G.,
Kemmer, K., El-Sayed, I., Augustyns, K., Neubauer, P.,
Wierenga, R.K. (2008) Structure-based protein engineering
efforts with a monomeric TIM variant: the importance of a
single point mutation for generating an active site with
suitable binding properties. PEDS 21:257-266.
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