Introduction
WP6 will experimentally validate mutation predictions made with the
SSP software and with BIOP's 3DM systems. The primary aim of these
experiments is validation of the in silico predictions to the extent
that the software and protocols can be improved using the validation
results. WP6 has as secondary aim the actual production of improved enzymes.
The partners in WP6 will aim at different aspects of experimental
validation. Partner EMAUG will provide its extensive knowledge in protein
engineering in general as support to all three experimental SMEs. ENZYM,
INGEN, and LEAD will use the SSP to tailor-design the proteins, which are
of key interests to them, hence partner ENZYM will concentrate on
transaminases and Baeyer-Villiger-monooxygenases (BVMO); partner INGEN
will focus on aminotransferases, amine oxidases, and carboxylic acid
reductases; while partner LEAD will base its in-house ongoing drug-design
related protein engineering efforts on SSP results and will provide
feedback on the outcome of those experiments.
Validation planning
EMAUG will use the bioinformatics tools available from the SSP and
from BIOP to further accelerate the development of enzymes for biocatalysis.
This will be performed for enzymes from the ~/~ hydrolase fold super-family
where they aim for the inter-conversion of enzyme activities, e.g. convert
an epoxide hydrolase into synthetically useful dehalogenases. Novel 3DM-systems
will be developed for different enzyme families to further expand the research
in this area to enable the identification of further useful transaminases,
transferases, BMVO, oxidases, and reductases with respect to substrate range,
enantioselectivity, and stability.
Thus, in close cooperation with partners 1, 3, and 5 targets for biocatalysis
will be identified for the different enzyme classes. Bioinformatics information
will be used by partners 2, 4, and 6 to design, create, and analyze mutant
libraries. The best hits thus identified experimentally in the laboratory
will be biochemically characterized to confirm predictions and their applicability
in biocatalysis. This will be performed in close collaboration between the
academic partner 2 and the experimental SMEs to take advantage of the
high-throughput screening facilities and the protein engineering knowledge at EMAUG.
Task 1
Enzymicals will use the protein engineering SSP to tailor-design its biocatalyst
on emerging issues. The primary focus will be on activity, selectivity and/or
stability of transaminases and Baeyer-Villiger monooxygenases. Secondary targets
are adjustments of the substrate spectra of representative catalysts from these
two classes. Predicted mutations are compared with results of other available
tools and selected positions are transferred to rational or evolutionary protein
design. The obtained experimental results will be used to validate and refine the
prediction tools and helps to understand the enzyme mechanisms. Improved variants
will be used for the company~s catalogue business and incorporated in the in-house
biocatalytic toolbox for the production of fine-chemicals.
Task 2
Ingenza will use the software tools available from the SSP and from BIOP
for two particular classes of experiments. First the focus will be on the
activity, selectivity, and thermostability of amino-transferase and carboxylic
acid reductase enzyme super-families. These experiments will mainly use the
multiple sequence alignment based tools. BIOP 3DM systems will be used to
generate the experimental designs. Partners 1, 3, and 6 will carefully analyse
the quality of these predictions and partner 3 will use this as input to a round
of improvement of the software (and perhaps the science) they use for the
production of 3DM systems. It takes partner BIOP days till weeks to produce
one 3DM system. This is too big an effort in terms of CPU usage to be made
freely available, so that EMBL's HSSP system (that is nowadays maintained by
partner CMBI) will be used on the SSP for the same multiple sequence based
purposes. HSSP alignments can be produced much faster than 3DM systems but
will be less accurate. Partners 1 and 3 will carefully analyse the quality
of HSSP based predictions and will try to improve the HSSP alignment system
further, if possible, and if improvements will not unreasonably increase the
CPU time efforts. The expected limitations of the predictions will be clearly
documented (in collaboration with partner 7).
Ingenza will (need to) stabilize a few of its target enzymes. For this
purpose they will use both the multiple sequence alignment based stability
prediction techniques that are part of BIOP's 3DM system, and they will use
the 'classical' protein structure and energy calculation based WHAT IF
methods that will be incorporated in the SSP. The experimental results will
be carefully analysed and these analyses will be made available to the users
of the SSP. The experimental results will obviously also be used to generate
ideas for better algorithms, protocols, or parameterisations for the prediction
of stabilizing mutations.
Task 3
LeadPharma performs protein engineering experiments in many of its
drug-design related projects. LeadPharma will not perform specially
designed experiments for the purpose of SSP validation, but they will
rather use the SSP as a normal tool in their in-house experimental design,
and they will, in close consultation with partner 7 (SAFAN) report back to
the NewProt team all its experiences. LeadPharma will be responsible for the
validation of the SSP in a pharmaceutical (drug design) context.
Iteration
Prediction and validation need to iterate continuously. WP6 will therefore
continuously communicate with WP2-5. It is envisaged that most exchange of
NewProt staff between the partner's labs will be related to this iteration
process.
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