Several new features have been developed:
A module that can collect reaction data (substrate, product) for the protein superfamily members.
A module that connects mutation data from literature data to Yasara.

Content: Discussion within the NewProt project: Selection of target compounds, wild-type enzymes and mutation strategies Describing the project objectives and discussion on the following topics:
A list of relevant target compounds containing chiral amino functions are presented and discussed
Chiral building blocks for these compounds are identified and categorized
Selection of concrete targets for investigation of reactivity with available ATAs
Presumed hindrance for conversion are the structure of the small binding pocket (SBP)
Expanding the substrate acceptance of small binding pocket as mutagenesis target
Selection of three concrete model substrate for investigations and screening procedures
Analytical methods are established
3DM based investigations yielded in a loop exchange strategy to expand the accessibility of the SBP of dedicated ATAs
This is our first mutation strategy
Generation of homology models with YASARA

Next tasks:
Investigation with available ATAs with model substrates and final selection of wt-enzymes
Validation of homology models with YASARA
Analysis of the sequence area which forms the SBP
Analysis of the models with HotSpot Wizard
Results will deliver a second mutation strategy
Generation of mutants for both strategies
Experimental validation of mutants Both partner will closely cooperate in molecular biology and biochemical work

• Major improvement of the 3DM website making 3DM usage more intuitive for protein engineers.
• Easy generation of sequence subsets
• Improved search algorithms
• Easy data comparisons between subsets
• Faster, interactive visualization of alignments
• Automated data transfer between yasara and 3DM:
  Structure selection directly from Yasara
  • Data visualization in relation to subsets
  • HUD data printing (e.g amino acid distributions)
• Easy switching between different 3DM options via an intuitive icon based structure
• Automated regeneration of sub 3DM systems
• Incorporation of enzyme reaction data (substrate, product, etc)
• New interactive network plotting algorithms for analysis of correlated mutations.

The aim of this study is to develop an (S)-ATA which can accept substrates with substituents on the ketone moiety larger than a methyl group. Analysis of the X-ray crystal structures of 4 enzymes (3HMU, 3I5T , 3GJU and 3FCR) identified 4 loops which could influence the substrate specificity of the enzymes. 1 of these 4 loops was chosen as a target for mutagenesis. A loop exchange strategy where the 'short' Loop 1 of 3I5T will be exchanged with the long Loop 1 of 3HMU (with 3HMU acting as the scaffold) is proposed. Thanks to the database, 3DM analysis of the amino acid distribution in the target Loop of 3HMU identified a highly conserved phenylalanine pair which should be considered in the design of further loop exchange mutants. CMA, using the 3DM tool Comulator, of both the 3HMU subfamily and the short loop 3I5T sequences revealed correlation between residues located in two different loops. This toll will also be used to guide further mutant design. Experimental verification and choice of substrates has been done and will be continued with close cooperation with project partner Enzymicals AG.

The Ingenza - Bio-Prodict contacts so far have been (reported by Ingenza):

1. Henk-Jan Joosten from Bio-Prodict visited Ingenza on April 25th for the first part of our formal training on the use of 3DM databases. Three Ingenza staff were trained (Reuben Carr, Franck Escalettes and myself) and a further 2 have since begun practising using the database.
2. Internal training using YASARA built-in training tools to gain expertise in protein molecular modelling.
3. Two 3DM systems have been supplied by Bio-Prodict (2-methylcitrate dehydratases, PLP-dependent transferases . Ingenza has begun to study the methyl citrate superfamily to identify suitable steis for mutagenesis. A third database based on tyorsine or phenylalanine ammonia lyase has been ordered.
4. We have cloned 3 enzymes from a thermophile which enable the chain elongation of alpha-keotacids (two are oxidoreductases). Two of the three proteins are active (by spectrophotometric and HPLC assay). The third is currently being tested. A database built around one sequence for the OR enzymes will be ordered. Specifically we will be attempting to increase the activity and oxygen stability of these proteins by comparison to stable enzymes within the superfamily. The activity of the cloned proteins is low at low temperature, we will try to identify enzymes with better activity at lower temperatures.

• The PMP models cannot be imported into the portal directly due to legal issues. Our solution is to make a button "find external models" that will show the PMP UI in a popup window. Here the user can find links to external models.
• The popup window will only show the graph with colored bars to indicate the coverage of the models and the table that shows the provider/template/sequence identity/coverage/etc of the models. So basically, all the model information that can already be found on the PMP site now. Juergen will help by making a separate website for the Newprot project.
• We will need to state very clearly in the text that the user can download these models and upload them all by themselves. In the freeware version we can add text about the good and automatic modelling options in YASARA that will become available when the user buys the full package.

• U.T. Bornscheuer: In silico Discovery and Application of Transaminases in Organic Synthesis
• E. Byström, H. Mallin, J. Muschiol. M. Höhne, U.T. Bornscheuer A novel mass transfer concept to facilitate enzyme-catalyzed reactions
• M. Höhne: Connecting unexplored protein crystal structures to enzymatic function: Identification of novel amine transaminases

• E. Champla, M. Patila, I.V. Pavlidis, H. Stamatis, M. Höhne, U.T. Bornscheuer: Efficient immobilization of -transaminases onto functionalized carbon-based nanomaterials
• H. Land, S. Chen, F. Steffen-Munsberg, A. Nobili, M. Hoöhne, U.T. Bornscheuer, P. Berglund: Semi-rational design to obtain a stable mutant of Chromobacterium violaceum amine transaminase
• C. Vickers, A. Mitin, A.V. Barro, H. Brundiek, A. Nobili, F. Steffen-Munsberg, M. Höhne, U.T. Bornscheuer: A strategy to enlarge the small binding pocket of (S)-selective amine transaminases
• U. Menyes: Synthesis of chiral building blocks and fine chemicals with unique enzymes

• Recently, the portal was updated to have the latest options available. One of the major new features is the visualization of the NPF files (e.g. computed from any of the included services) as a YASARA scene file. Currently the option to create and download the scene is located at the NPF-file page. Andreas will move the “create” and “download-option” to the structure page and combine them in one button.
• Andreas will make sure that all the generated YASARA-scenes have unique and descriptive names.
• The YASARA-scene created for ProlineMutation-values does not show the correct results yet, probably caused by the -999,99 values in the output. These values should be ignored as this is WHAT IF’s way to indicate values that could not be calculated. However, the value lies within the range of -1000 to 1000. Hanka will contact Maarten or Gert to see how we can change the range.
• Gert is going to work on new services that can be incorporated in the Portal soon. Now the framework is ready it should be easy to add new services.

• The HotSpotWizard had some small problems which are currently solved. It also changed its port which caused some firewall problems for the NewProt Portal. Andreas will discuss this with their system administrator soon.
• PMP is still showing both the existing structures and models on the same page. Juergen did not reply on the emails yet. Hanka will send another email by the end of next week.

• The WHAT IF-webservice output will be shown in a large table. We expect to have many different servers and therefore we need to think of a way to visualize the data in a smart and informative way. Andreas and Peter will soon discuss the possibilities here. Options to think about: one large scrollable table, one large table that can be costumized on demand by the user using small collapse-buttons, movable collums, or a system that allows the user to create his/her own table from the available data.
• Hanka will create some new icons for the delete and archive buttons, and help
• Andreas will add the option to archive projects directly on the user-welcome page.
• The Workbench has an option to add a help-function. This is a little icon that will cause a pop-up with a helpful text. Hanka will think about the location and text-for these help-buttons.