In biocatalysis, structural knowledge regarding an enzyme and its substrate interactions complements and guides experimental investigations. Structural knowledge regarding an enzyme or a biocatalytic reaction system can be generated through computational techniques, such as homology- or molecular modeling. For this type of computational work, a computer program developed for molecular modeling of proteins is required. Here, we describe the use of the program YASARA Structure. Protocols for two specific biocatalytic applications, including both homology modeling and molecular modeling such as energy minimization, molecular docking simulations and molecular dynamics simulations, are shown. The applications are chosen to give realistic examples showing how structural knowledge through homology and molecular modeling is used to guide biocatalytic investigations and protein engineering studies.
The bacterial ω‐transaminase from Chromobacterium violaceum (Cv‐ωTA, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/6/1/18.html) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5′‐phosphate (PLP). Here, we present four crystal structures of Cv‐ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo‐Cv‐ωTA structure reveals unique ‘relaxed’ conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered ‘roof ’ over the PLP‐binding site, whereas in the other apo form and the holo form the ‘roof’ is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the ‘roof’ structure may be associated with substrate binding in Cv‐ωTA and some other transaminases. Database The atomic coordinates and structure factors for the Chromobacterium violaceumω‐transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6U for the holoenzyme, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6R for the apo1 form, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6T for the apo2 form and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A72 for the mixed form Structured digital abstract http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0038 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300874) http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300763) http://www.uniprot.org/uniprot/Q7NWG4 and http://www.uniprot.org/uniprot/Q7NWG4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8300950)
One lock for different keys: A flexible arginine in the active site allows γ‐aminobutyrate:pyruvate transaminases to bind the chemically different substrates L‐alanine and γ‐aminobutyric acid. Moreover, a flexible arginine residue facilitates the promiscuous conversion of (S)‐amines and ketones. The degree of promiscuity can be related to distinct key amino acids lying at the surface of the active site.
Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio‐, chemo‐ and stereoselective transformations. Enzyme promiscuity allows optimisation of industrial processes that require reaction conditions different from those in nature. Many enzymes can be used in reactions completely different from the reaction the enzyme originally evolved to perform. Such catalytically promiscuous reactions can be secondary activities hidden behind a native activity and might be discovered either in screening for that particular activity or, alternatively, by chance. Recently, researchers have designed enzymes to show catalytic promiscuity. It is also possible to design new enzymes from scratch by computer modelling (de novo design), but most work published to date starts from a known enzyme backbone. Promiscuous activity might also be induced or enhanced by rational design or directed evolution (or combinations thereof). Enzyme catalytic promiscuity provides fundamental knowledge about enzyme/substrate interactions and the evolution of new enzymes. New enzymes are required by industry, which needs to optimise chemical processes in an environmentally sustainable way. In this review various aspects of enzyme catalytic promiscuity are considered from a biocatalytic perspective.
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