Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Abstract

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system in studies of the role of protein dynamics in catalysis, but so far these studies were limited by the availability of structural information. Second, CbFDH and its mutants are of use in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in its commercial development. Here, we report the crystallization and structural determination of both holo-CbFDH and apo-CbFDH. The free energy barrier for the catalyzed reaction is computed, and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: The apo-enzyme crystal was obtained under co-crystallization conditions with a saturating concentration of both the cofactor (NAD+) and inhibitor (azide), which has a nM dissociation constant. It was found that the fraction of the apo-enzyme present in the solution is less than 1.7x10−7 (i.e. the solution is 99.9999% holo-enzyme). This is an extreme case where the crystal structure represents an insignificant fraction of enzyme in solution, and a mechanism rationalizing this phenomenon is presented.

Protein Mass Effects on Formate Dehydrogenase

“…All enzymes were purified to homogeneity in buffered H 2 O using affinity chromatography according to methods described elsewhere. 41,52,53 Monomeric molecular weights were determined by ESI-MS after purity check with SDS-PAGE. In addition to the intact mass, the ESI-MS of the heavy FDH treated with 8M urea (1 hr at room temperature) was also determined in order to evaluate the extent of non-exchanged labile hydrogen atoms.…”

“…(A) Arrhenius plot of forward commitment on V/K (C f ) for light (green, from ref 41 ) half-heavy (red) and heavy (blue) FDHs. (B) ΔE a of C f vs. inverse square root of protein mass.…”

“…FDH is a homodimer with a total mass of 80,620 Da. 39-41 In the reaction catalyzed by FDH, a hydride is transferred from formate ion to the C-4 position of NAD + to generate NADH and CO 2 , as illustrated in Scheme 1. 42 …”

“…In the context of this paper, the temperature dependence of KIEs accounts for the nature of the TRS for the FDH catalyzed C-H→C hydride transfer. 41,43 …”

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy

“…Those experiments used a commercial FDH extracted from yeast ( Candida boidinii ), which we recently found to be a mixture of different isozymes with different masses and different charges. 9 That mixture likely led to heterogeneous structures and dynamics whose average further masked the true nature of the FFCF. In the current report we use only one of these isoenzymes produced as described elsewhere.…”