Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.
The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was 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 apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Qi
Q
Guo
Protein Crystallography Facility and Department of Biochemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Lokesh
L
Gakhar
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Kyle
K
Wickersham
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Kevin
K
Francis
Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University , Ramat-Gan 5290002, Israel.
Alexandra
A
Vardi-Kilshtain
Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University , Ramat-Gan 5290002, Israel.
Dan T
DT
Major
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Christopher M
CM
Cheatum
Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Amnon
A
Kohen
D001051:Apoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D005560:Formate Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D019810:Sodium Azide / Q000737:chemistry
D001051:Apoenzymes; D005656:Fungal Proteins; D009243:NAD; D019810:Sodium Azide; D005560:Formate Dehydrogenases