One hotly debated topic in enzymology is whether and how the fast protein structural fluctuations affect the catalyzed chemistry in enzymes' active site. Formate dehydrogenase from Candida boidinii (CbFDH) was found to be a unique model system to address this question. Our previous kinetic and 2DIR studies identified probable coupling between femtosecond-picosecond dynamics and the catalyzed H-tunneling step. Structural information is critical for mutational studies that may reveal direct correlation between dynamics and the catalyzed hydride transfer. Here, we report crystallization and structural solution of both holo-CbFDH (with NAD+ and azide)  and the apo-CbFDH using the recombinant CbFDH. The activity of this recombinant enzyme is identical to that of the commercial CbFDH previously used. Surprisingly the apo-enzyme has also been crystallized under saturation of NAD+ and azide, which means the majority of complexes in solution are excluded during the crystallization step. The new CbFDH structures were compared to those of Pseudomonas sp. 101 FDH (PsFDH), and the mutant holo-CbFDH. The special alignment between CbFDH and PsFDH was excellent for both closed and open conformations for holo- and apo-enzymes relatively. The intrinsic kinetic isotope effects (KIEs) and their temperature dependency for the recombinant and the commercial mixture was found to be very similar, which opens the door to mutational studies of the recombinant enzyme in pursuit of correlation between the fast dynamics and the nature of hydride transfer.1. Bandaria, J. N.; Cheatum, C. M.; Kohen, A., J. Am. Chem. Soc. 2009,  131 (29), 10151-10155.2. Lamzin, V. S.; Dauter, Z.; Popov, V. O.; Harutyunyan, E. H.; Wilson, K. S., J. Mol. Biol. 1994,  236 (3), 759-85.3. Schirwitz, K.; Schmidt, A.; Lamzin, V. S., Protein Sci. 2007,  16 (6), 1146-56.

Structural and kinetic studies of formate dehydrogenase

248th National Meeting (2014)

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