Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Qi Guo; Lokesh Gakhar; Kyle Wickersham; Kevin Francis; Alexandra Vardi-Kilshtain; Dan T Major; Christopher M Cheatum; Amnon Kohen

doi:10.1021/acs.biochem.6b00181

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Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

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Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii

Qi Guo, Lokesh Gakhar, Kyle Wickersham, Kevin Francis, Alexandra Vardi-Kilshtain, Dan T Major, Christopher M Cheatum and Amnon Kohen

Biochemistry (Easton), Vol.55(19), pp.2760-2771

05/17/2016

DOI: 10.1021/acs.biochem.6b00181

PMCID: PMC4917879

PMID: 27100912

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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 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.

Fungal Proteins - chemistry

Candida - genetics

Formate Dehydrogenases - metabolism

Candida - enzymology

Fungal Proteins - antagonists & inhibitors

Fungal Proteins - genetics

Formate Dehydrogenases - genetics

NAD - chemistry

Formate Dehydrogenases - chemistry

Apoenzymes - genetics

Sodium Azide - chemistry

Apoenzymes - metabolism

Apoenzymes - chemistry

Formate Dehydrogenases - antagonists & inhibitors

Kinetics

Apoenzymes - antagonists & inhibitors

NAD - metabolism

Fungal Proteins - metabolism

Details

Title: Subtitle: Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii
Creators: Qi Guo - Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States
Lokesh Gakhar - Protein Crystallography Facility and Department of Biochemistry, University of Iowa , Iowa City, Iowa 52242, United States
Kyle Wickersham - Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States
Kevin Francis - Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States
Alexandra Vardi-Kilshtain - Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University , Ramat-Gan 5290002, Israel
Dan T Major - Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University , Ramat-Gan 5290002, Israel
Christopher M Cheatum - Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States
Amnon Kohen - Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.55(19), pp.2760-2771
DOI: 10.1021/acs.biochem.6b00181
PMID: 27100912
PMCID: PMC4917879
NLM abbreviation: Biochemistry
ISSN: 1520-4995
eISSN: 1520-4995
Publisher: United States
Grant note: R01 GM079368 / NIGMS NIH HHS R01 GM065368 / NIGMS NIH HHS
Language: English
Date published: 05/17/2016
Academic Unit: Core Research Facilities; Biochemistry and Molecular Biology; Chemistry; Medicine Administration
Record Identifier: 9983985964302771

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