Subunit Arrangement of a 2-Ketoisovalerate Ferredoxin Oxidoreductase from Thermococcus profundus Revealed by a Low Resolution X-Ray Analysis

Yukiko Ozawa¹, Yasufumi Umena², Takeo Imai¹, Yukio Morimoto^3*

¹Department of Life Science and Graduate School of Life Science, Rikkyo (St. Paul’s) University, Tokyo, Japan.
²Photosynthesis Research Center, Okayama University, Okayama, Japan.
³Research Reactor Institute, Kyoto University, Osaka, Japan.

DOI: 10.4236/aer.2015.33008                

Abstract

2-ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of aliphatic amino acid-derived 2-keto acids. The enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (αβγδ)2 consisting of four different subunits, α = 45 kDa, β = 31 kDa, γ = 22 kDa and δ = 13 kDa, respectively, and it has three [4Fe-4S] clusters per αβγδ-protomer, similar to other ferredoxin oxidoreductases. In the present study, the native enzyme was purified from this strain and crystallized to give rod-like crystals that were suitable for X-ray diffraction experiments. The crystals belonged to space group P41212, with unit-cell parameters a = b = 136.20 Å, c = 221.07 Å. Diffraction images were processed to a resolution of 3.0 Å. The data collected so far indicate the approximate molecular boundaries and a partial main-chain trace of the enzyme.

Keywords

Oxidoreductase, X-Ray Analysis, Iron-Sulfur Cluster

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Ma, K., Loessner, H.J., Heider, J., Johnson, M.K. and Adams, M.W.W. (1995) Effects of Elemental Sulfur on the Metabolism of the Deep-Sea Hyperthermophilic Archaeon Thermococcus Strain ES-1: Characterization of a Sulfur-Regulated, Non-Heme Iron Alcohol Dehydrogenase. Journal of Bacteriology, 177, 4748-4756.
[2]	Heider, J., Ma, K. and Adamas, M.W.W. (1995) Purification, Characterization, and Metabolic Function of Tungsten- Containing Aldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic and Proteolytic Archaeon Thermococcus Strain ES-1. Journal of Bacteriology, 177, 4757-4764.
[3]	Adams, M.W.W. and Kletzin, A. (1996) In: Adams, M.W.W., Ed., Advances in Protein Chemistry, Vol. 48, Academic Press, New York, 101-180.
[4]	Chabriere, E., Charon, M.-H., Volbeda, A., Pieulle, L., Hatchikian, E.C. and Fontecilla-Camps, J.C. (1999) Crystal Structures of the Key Anaerobic Enzyme Pyruvate: Ferredoxin Oxidoreductase, Free and in Complex with Pyruvate. Nature Structural Biology, 6, 182-189. http://dx.doi.org/10.1038/5870
[5]	Ragsdale, S.W. (2003) Pyruvate Ferredoxin Oxidoreductase and Its Radical Intermediate. Chemical Reviews, 103, 2333-2346. http://dx.doi.org/10.1021/cr020423e
[6]	Ma, K., Hutchins, A., Sung, S.J. and Adams, M.W.W. (1997) Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon, Pyrococcus furiosus, Functions as a CoA-Dependent Pyruvate Decarboxylase. Proceedings of the National Academy of Sciences of the United States of America, 94, 9608-9613. http://dx.doi.org/10.1073/pnas.94.18.9608
[7]	Mai, X. and Adams, M.W.W. (1994) Indolepyruvate Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus. A New Enzyme Involved in Peptide Fermentation. Journal of Biological Chemistry, 269, 16726-16732.
[8]	Mai, X. and Adams, M.W.W. (1996) Characterization of a Fourth Type of 2-Keto Acid-Oxidizing Enzyme from a Hyperthermophilic Archaeon: 2-Ketoglutarate Ferredoxin Oxidoreductase from Thermococcus litoralis. Journal of Bacteriology, 178, 5890-5896.
[9]	Heider, J., Mai, X. and Adams, M.W.W. (1996) Characterization of 2-Ketoisovalerate Ferredoxin Oxidoreductase, a New and Reversible Coenzyme A-Dependent Enzyme Involved in Peptide Fermentation by Hyperthermophilic Archaea. Journal of Bacteriology, 178, 780-787.
[10]	Tersteegen, A., Linder, D., Thauer, R. and Hedderich, R. (1997) Structures and Functions of Four Anabolic 2-Oxoacid Oxidoreductases in Methanobacterium Thermoautotrophicum. European Journal of Biochemistry, 244, 862-868. http://dx.doi.org/10.1111/j.1432-1033.1997.00862.x
[11]	Siddiqui, M.A., Fujiwara, S. and Imanaka, T. (1997) Indolepyruvate Ferredoxin Oxidoreductase from Pyrococcus ap. KOD1 Possesses a Mosaic Structure Showing Features of Various Oxidoreductases. Molecular and General Genetics, 254, 433-439.
[12]	Ozawa, Y., Nakamura, T., Kamata, N., Yasujima, D., Urushiyama, A., Yamakura, F., Ohmori, D. and Imai, T. (2005) Thermococcus profundus 2-Ketoisovalerate Ferredoxin Oxidoreductase, a Key Enzyme in the Archaeal Energy-Producing Amino Acid Metabolic Pathway. The Journal of Biochemistry, 137, 101-107. http://dx.doi.org/10.1093/jb/mvi012
[13]	Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[14]	Battye, T.G.G., Kontogiannis, L., Johnson, O., Powell, H.R. and Leslie, A.G.W. (2011) iMOSFLM: A New Graphical Interface for Diffraction-Image Processing with MOSFLM. Acta Crystallographica, D67, 271-281. http://dx.doi.org/10.1107/s0907444910048675
[15]	Evans, P. (2006) Scaling and Assessment of Data Quality. Acta Crystallographica, D62, 72-82. http://dx.doi.org/10.1107/s0907444905036693
[16]	Potterton, E., Briggs, P., Turkenburg, M. and Dodson, E. (2003) A Graphical User Interface to the CCP4 Program Suite. Acta Crystallographica, D59, 1131-1137. http://dx.doi.org/10.1107/S0907444903008126
[17]	Matthews, B.W. (1968) Solvent Content of Protein Crystals. Journal of Molecular Biology, 33, 491-497. http://dx.doi.org/10.1016/0022-2836(68)90205-2
[18]	Vagin, A. and Teplyakov, A. (2010) Molecular Replacement with MOLREP. Acta Crystallographica, D66, 22-25. http://dx.doi.org/10.1107/S0907444909042589
[19]	Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C. and Zwart, P.H. (2010) PHENIX: A Comprehensive Python-Based System for Macromolecular Structure Solution. Acta Crystallographica, D66, 213-221. http://dx.doi.org/10.1107/s0907444909052925