Using Neutron Scattering, Diffraction, and Computational Methods to Identify the Molecular Determinants that Govern Pyridoxal 5’-Phosphate-Dependent Catalysis

Orateur : Steven DAJNOWICZ (Oak Ridge National Laboratory)

Pyridoxal-5’-phosphate (PLP, vitamin B6) is one of the most ubiquitous cofactors in biological systems. PLP-dependent enzymes are essential proteins that catalyze a diverse number of reactions : transamination, racemization, phosphorylation, decarboxylation, aldol cleavage, elimination, and replacement. At least five different fold types comprise the PLP-dependent families of enzymes, each catalyzing a specific type of chemistry. In efforts aimed at developing medicinal and protein engineering applications, researchers have tried for many years to elucidate the underlying physical details that contribute to the reaction specificity in different PLP-dependent enzymes. Our current hypothesis is that active site interactions modulate the electronics within PLP-cofactor to optimize the desired chemical reaction in each family of enzymes. One way to modulate the electronics is to invoke different protonation states of PLP-substrate complex. Moreover, the active site local environment can promote the different protonation profiles of PLP during the catalytic cycle. Here, we report the first neutron crystal structure of a PLP dependent enzyme, aspartate aminotransferase (AAT). Neutron protein crystallography is one of the most powerful techniques to study the positions of hydrogen/deuterium atoms in a macromolecule. The AAT crystal was soaked with α-methyl-DL-aspartic acid (substrate derivative) to capture AAT in both the ground (internal aldimine) and substrate bound (external aldimine) states. Interestingly, the neutron structure of AAT shows that PLP and active site residues have different protonation states in the ground vs. substrate bound states. In the efforts to validate our hypothesis, an interdisciplinary approach using neutron/X-ray diffraction, inelastic neutron scattering, and molecular simulations has been implemented to the identify the molecular determinants that govern catalysis.

Télécharger dans mon agenda