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|Title: ||Mechanisms of silicate polymerisation, carbohydrate epimerisation and metalloprotease inhibition|
|Authors: ||Kowatz, Thomas|
|Supervisors: ||Naismith, James|
|Issue Date: ||30-Nov-2009|
|Abstract: ||In biotechnology and drug delivery silica materials are of interest but the controlled generation of silicic acid is difficult. To get more insight into the molecular mechanisms that control biosilification, it is important to study the proteins involved in this process. The sponge protein silicatein α synthesises part of the axial filament in the spicules which in situ polymerises silicic acid. It has been demonstrated that the polymerisation of siloxanes such as for example tetraethoxysilane (TEOS) can be carried out by both wild type and recombinant silicatein α. Unfortunately, it has not been possible yet to get reasonable amounts of wild type or recombinant silicatein α to perform biophysical studies. The human cysteine protease cathepsin L has almost 50 % identical residues with silicatein α. To get more insight into the mechanism of silica polymerisation, cathepsin L mutants were generated by our collaborators. Those mutants show sequence features and activity specific for silicatein α. The X-ray structure of one of those mutants (mutant 4SER) to 1.5 Å has allowed us to propose a new chemical mechanism for the catalysis of silicic acid polymerisation.
ADP-β-D-glycero-D-mannoheptose and ADP-β-L-glycero-D-mannoheptose are
interconverted by the SDR-enzyme ADP-β-L-glycero-D-mannoheptose 6-epimerase
(AGME). This epimerisation reaction is the final reaction in the biosynthetic route of
the precursor of heptose. Heptose is a part of the inner core of the lipopolysaccharide
in Gram-negative bacteria. In mutants which do not have heptose, nonpolar
compounds can penetrate more easily through the outer membrane. These mutants
also show less pathogenicity. As a consequence the lipopolysaccharide biosynthetic
pathway represents an interesting target for antimicrobial compounds. The crystal
structure of AGME in complex with ADP-α-glucose has already been solved.
From this structure a catalytic mechanism for this enzyme has been proposed with
Tyr140 and Lys178 operating as acid/base residues.
The disordered nature of the nucleotide sugar’s glucose moiety in the previous
structure due to the wrong configuration of the sugar has hindered assignment of a
mechanism. The determination of the X-ray structure of AGME Y140F in complex
with a substrate in the β-manno configuration (ADP-β-mannose) to 2.4 Å resolution
has given new insight into the mechanism of this SDR enzyme. A mechanism is
proposed with only Tyr140 operating as catalytic acid/base.
Initially it was thought that MMP-3 participates in the synovitis cascade.
Glycoproteins, several parts of the ECM such as fibronectin and laminin and also
collagens and procollagens are targets of this matrixin. Furthermore MMP-3 can
undergo autocatalysis and is also able to cleave a range of other members of the
matrixin family. Matrixins also play an important role in diseases such as cancer,
rheumatoid arthritis and osteoporosis. This makes them targets for inhibitor design.
Many structures of matrix metalloproteinases, such as stromelysin-1, in complex with
various inhibitors have already been solved. The structures of the catalytic domain of
MMP-3 in complex with two nonpeptide inhibitors are discussed.|
|Other Identifiers: ||uk.bl.ethos.552299|
|Publisher: ||University of St Andrews|
|Appears in Collections:||Chemistry Theses|
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