Work Package 4
Enzyme Design: Enzyme Crystallization and Bioinformatics
Structure determination of (S) and (R)-selective ω-transaminases by protein X-ray crystallography
Niels van Oosterwijk, Johan Hekelaar and Bauke W. Dijkstra
University of Groningen, The Netherlands
In work package 4 we determine protein X-ray crystal structures of (S) and (R)-selective ω-transaminases identified and characterized in the other WPs. Crystal structures of these enzymes can be used to obtain highly detailed insights into the functioning of the enzymes and subsequently may allow the creation of improved designer mutants by molecular engineering.
Several pyridoxal-phosphate (PLP) dependent (S)-selective transaminases and one (R)-selective ω-transaminase were selected for crystallization. Elucidation of the three-dimensional structures of these ω-transaminases starts with their purification and crystallization. The obtained crystals (Fig 1. A) are subjected to X-ray diffraction analysis. From these experiments their structure can be solved (example in Fig 1 B).
Fig 1. A) Typical crystals obtained for the ω-transaminases. B) Cartoon representation of the four ω-transaminase molecules found in the asymmetric unit of the crystal. The green and red molecules form a tight dimer, like the blue and yellow molecules.
The structures will be used to analyze substrate binding and the stereo-selectivity of the selected (R)-selective ω-transaminases. Currently only a few (R)-selective ω-transaminases are known and 3D-structures are still lacking. An X-ray structure of a (R)-selective ω-transaminase will allow us to study its substrate specificity and the structural determinants for stereo-specificity.