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.

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 (V_M) [17] is calculated to be 2.2 ųDa⁻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 R_free 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.

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.

YukikoOzawa,YasufumiUmena,TakeoImai,YukioMorimoto, (2015) Subunit Arrangement of a 2-Ketoisovalerate Ferredoxin Oxidoreductase from Thermococcus profundus Revealed by a Low Resolution X-Ray Analysis. Advances in Enzyme Research,03,75-80. doi: 10.4236/aer.2015.33008