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


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.

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Ozawa, Y. , Umena, Y. , Imai, T. and Morimoto, Y. (2015) Subunit Arrangement of a 2-Ketoisovalerate Ferredoxin Oxidoreductase from Thermococcus profundus Revealed by a Low Resolution X-Ray Analysis. Advances in Enzyme Research, 3, 75-80. doi: 10.4236/aer.2015.33008.

1. Introduction

An energy-producing pathway in the hyperthermophilic archaeon, Thermococcus profundus, has been proposed to degrade amino acids [1] [2] . In this pathway, amino acids produced from some peptides by peptidases are converted to their corresponding 2-keto acids by transaminases. The 2-ketoisovalerate ferredoxin oxidoreductase (VOR) catalyzes a reaction to produce acetyl CoA in the presence of coenzyme A from these 2-keto acids through oxidative decarboxylation. Pyruvate ferredoxin oxidoreductase (PFOR) is one of the well-characterized members of the ferredoxin-dependent enzyme family [3] - [5] , and it has been shown to catalyze the production of acetaldehyde in the presence of coenzyme A [6] . However, the enzymes indolepyruvate ferredoxin oxidoreductase (IOR) [7] , 2-ketoglutarate ferredoxin oxidoreductase (KGOR) [8] and 2-ketoisovalerate ferredoxin oxidoreductase (VOR) [9] are relatively poorly characterized, and their physiological functions have not been established experimentally [10] [11] . Four types of enzyme form a structurally related superfamily in which the enzymes each contain thiamine pyrophosphate, magnesium ions, and one to three [4Fe-4S] cluster(s) as prosthetic groups. The spatial arrangement of the cofactors in D. africanus PFOR is clarified by X-ray crystallographic analysis [4] , but the three-dimensional structures of the other three types of enzyme are not yet known. In order to investigate the structure-function relationships and deduce specific substrate recognition mechanisms of these four types of enzyme (PFOR, VOR, KGOR, IOR), we report here the purification and crystallization of VOR from Thermococcus profundus and a preliminary analysis of crystallographic data collected for this enzyme.

2. Materials and Methods

2.1. Isolation and Purification of Protein

The protein was purified from the native organism, Thermococcus profundus. Isolation of the VOR has been described in a previous report [12] . Cell-free extracts were loaded onto a Q-sepharose Fast Flow (GE Healthcare) column equilibrated with buffer. After a wash step the enzyme fraction was eluted with a NaCl linear gradient and then applied onto a hydroxyapatite column followed by a RedTOYOPEALE (TOSOH Bioscience) column. The fractions that contained VOR alone (Figure 1(a)) in the buffer of 50 mM Tris-HCl, pH 8, 1 mM thiamine pyrophosphate, 1 mM MgSO4, 0.5 M NaCl, 2 mM dithiothreitol, 2 mM sodium dithionite (SIGMA Aldrich) were concentrated with an Amicon YM50 (Merck Millipore Corporation) membrane, in which the buffer was degassed and purged oxygen by a babbling of Ar gas. Protein concentration was estimated by the method of Bradford [13] modified by use of the rose bengal (Food Red No. 105) [12] .

(a) (b)

Figure 1. Purity and crystals of VOR. (a) Silver-stained SDS polyacrylamide gels showing the proteins within one dissolved crystal; left lane, molecular weight marker; middle lane, crystal 1; right lane crystal 2 (high contrast). A line is drawn between lanes showing proteins in crystal 1 and crystal 2; (b) Brownish crystals with a few stains caused by protein precipitation in a cryo-loop at the goniometer head. Scale bar is 0.1 mm.

2.2. Crystallization

Crystallization of VOR was carried out using a hanging-drop vapor diffusion method at 293 K under the following condition: 2 μl protein solution concentrated to 10 mg∙ml−1 was mixed with 2 μl reservoir solution containing 13% (w/v) PEG6000, 0.1 M sodium cacodylate buffer at pH 6.0, 0.1 M MgCl2, 2 mM sodium dithionite, 10 mM dithiothreitol and the droplet was equilibrated against 1 ml reservoir solution (Table 1). These crystallization handling was set up in an anaerobic chamber to keep oxygen free with nitrogen gas flow. A few weeks later, crystals appeared and grew to rod-shaped crystals of 0.1 mm length (Figure 1(b)).

2.3. Data Collection and Processing

Crystals of VOR were soaked for approximately 20 - 30 sec in a cryoprotectant solution, which was the reservoir solution containing 10% - 20% glycerol. The crystals were mounted and flash-cooled at 100 K. Diffraction data were collected using synchrotron radiation. The diffraction data, however, were not of sufficient quality to proceed under 15% glycerol condition, because of the low resolution (8 Å) and the splitting of diffraction spots. Since crystals treated without a cryoprotectant solution diffracted well to higher resolution (4 Å), data acquisition for a decision of cell parameters was carried out by enclosing the crystal in a quartz capillary with a small amount of the buffer at 288 K. The crystal was analyzed on the BL5A at the Photon Factory (Tsukuba, Japan) and the BL44XU in the Spring-8 (Harima, Japan). After decision of cell parameters and crystal setting, a crystal treated by 20% glycerol protectant and frozen by dipping it into liquid N2 was used for high and low data acquisition. The crystal-to-detector distance was 800mm for low resolution (116.0 - 5.1 Å) and 500 mm for high resolution (50.0 - 3.0 Å) data, and exposure times were 5 and 3 seconds, respectively. The data were collected from 1 to 180 degrees with an oscillation range of 0.5 degree (low resolution) and 1 degree (high resolution). All diffraction images were processed with the programs iMOSFLM [14] and SCALA [15] of the CCP4 program suite [16] . All data from high and low resolution data sets were merged and scaled to one data set up to 3 Å resolution. The statistics of data collection and processing are summarized in Table 2 and Table 3.

Table 1. Crystallization condition and details.

Table 2. Crystallographic data and collection.

aValues in parentheses are for the highest-resolution shell.

Table 3. Refinement statistics.

bRwork was calculated from the working set (95% of the data). cRfree was calculated from the test set (5% of the data).

3. Results and Discussion

The VOR crystal is a rectangular prism, crystallizing in a tetragonal crystal group with unit-cell parameters: a = b = 136.20 Å, c = 221.07 Å. The space group, however, is uncertain because the reflection data characterizing the extinctions along the a* = b* and c* axes are not recorded. A silver stained SDS electrophoresis gel of one dissolved crystal clearly showed four bands demonstrating that all four subunits were present in the crystal (Figure 1(a)). The calculated molecular weight of the VOR αβγδ-protomer is 111 kDa, and the Matthews coefficient (VM) [17] is calculated to be 2.2 Å3Da−1 for sixteen molecules per unit cell. A self-rotation function calculated using low resolution data (4 Å) from the P1 space group shows definite peaks at each of the a-, b-, and c-axes and a diagonal position between a- and b- for a 2-fold symmetry with kappa = 180˚, and also one peak along the c-axis for a 4-fold symmetry with kappa = 90˚. From these results, we have concluded that the crystal corresponds to the 4/mmm Laue group and two αβγδ-protomers exist in each asymmetric unit.

Although the amino acid sequence of the VOR from T. profundus has not been determined, the sequence of a homologous enzyme, VOR from P. furiosus, has been determined and amino acid identities are 29% (pfVOR: 299 - 664), 33% (792 - 990), 23% (2 - 288) (Figure 2 upper) and 27% (699 - 747) (Figure 2 lower) against the PFOR (PDB entry 1b0p, [4] ) from D. africanus. We tried to solve main-chain structures of the VOR; the PFOR was used as a model molecule for a preliminary structural analysis. A main-chain model created from the PFOR coordinates by deleting side-chain atoms of its amino acids is available for use in a molecular replacement method (MOLREP [18] ), and searches of the unit cell are carried out using this model for several related space groups. A proper solution was obtained for the space group P41212 with values of 0.234 (Score) and 2.038 (Contrast). A preliminary structural analysis has been carried out using the program Phenix [19] and employing this space group and the PFOR main-chain model. PFOR consists of a single polypeptide chain composed of 1232 amino acids, and it appears to have four segments likely to be four domains (PFOR: 2 - 416; pfVOR: α-subunit, 417 - 630: δ, 631 - 786: γ, 787 - 1179: β) from its N- to C-terminus. The VOR structure, which is composed of four subunits (α, β, γ, δ, Figure 1(a)), is modified by rigid-body refinement and auto-model building using the Phenix program, with initial phases obtained from the low resolution data, producing an electron density map showing boundaries of the VOR molecule in the unit cell and assigning four subunits (Figure 3). However, the secondary structures of the chain were not yet well defined. The current R-factor and Rfree are 0.3196 and 0.3674 (Table 3), respectively. Searches for positions of the iron-sulfur clusters in the δ and β-subunits corresponding to the PFOR structure and of the other protomer in an asymmetric unit, and modifications of the VOR model corresponding to molecular weight of each subunit using a solvent-flattening method to improve the initial phases, are currently in progress.

Figure 2. Amino acid alignment of the pfVOR and PFOR. Cys bound Fe atoms are bold. Upper: pfVOR γδ-subunit, lower: a part of β-subunit.

(a) (b)

Figure 3. Electron density maps of the VOR structure at 3 Å resolution; (a) boundaries of the enzyme molecules, (b) subunit arrangement of the enzyme (α-subunit: yellow, β: magenta, γ: cyan, δ: green).


This work was supported by a Grants-in-Aid for Scientific Research (22570113(YM)) from the Ministry of Education, Science and Culture of Japan and the Customer Care Plan Foundation (YM), and by a Project Research Program under approvals 25P6-1 and 23P2-5 of Research Reactor Institute, Kyoto University. The synchrotron radiation experiments were conducted under the approvals 2006AB1422 and 2011AB6647 of SPring-8 and 2006G180 of PF.


*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.


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