**4. Crystal structure of ICDH**

320 Current Trends in X-Ray Crystallography

Thyroglobulin Ferritin BSA

1000 kDa

100

Cymotrypsinogen

SOD

Ovalbumin

10

Fig. 7. HPLC gel permeation profiles of *Tth* ICDH solution with and without the thick diamond-shaped crystals (crystal form II). (a) HPLC gel permeation profile of *Tth* ICDH being incubated under the crystallization condition for crystal form II; (b) that of intact *Tth* ICDH. The peaks numbered 1, 2, 3, and 4 correspond to about 400 k, about 300 k, about 220

Elution time (min)

0 5 10 15 20 25 30

(monomer) molecules, respectively. Since protein crystals usually include solvent molecules at high content it is difficult to speculate how many *Tth* ICDH molecules constitute the one ellipsoidal body. Taking these results obtained from AFM imaging and HPLC gel filtration into account, one can infer that *Tth* ICDH crystal form II should be comprised of oligomeric building blocks piled one on top of another. The building unit is most likely an octamer (4 dimers), and the next likely to be a hexamer (3 dimers) from the HPLC profile (Fig. 7), where both are made of *Tth* ICDH dimer as a basic unit. In crystal form II of *Tth* ICDH the exact arrangement and manner of the formation are still obscure, although there is possibility that *Tth* ICDH supramolecular complex acts as a block that interacts together in the process of spontaneous building up of form II crystals under the favorable crystallization condition described above. Needless to say, in order to determine a crystal structure of a certain protein species by X-ray method, crystals that well diffract X-rays to high resolution, and at the same time, that contain possibly small number of molecules in crystallographic asymmetric unit are prerequisite (N. Ishii et al., 2000a, 2000b; Shimamura et al., 2004). On the other hand, to construct some architectures in the nano-scale using protein molecules as building blocks we have to understand the nature of interactions between protein molecules, namely, how the

k, and about 98 kDa respectively.

Absorbance

0.008

(a)

(b)

ICDH evolved early and is widely distributed among archaea, bacteria, and eukarya. Such an evolutional trace can be found in diverse primary structures, various oligomeric forms taken, and different specificity as to cofactors (Steen et al., 2001). It has been proposed that NAD+-specific ICDH may be an ancestor enzyme that functions in CO2 fixation in an early stage of evolution of the Krebs cycle (Shiba et al., 1985).

We have unexpectedly obtained the crystals (form II) of the supramolecular complex of *Tth* ICDH and concentrated on surveying how these building block molecules pile up and selfassemblize into the crystal form II. Finally we have revealed the mechanism of the hierarchical formation that *Tth* ICDH molecules reside, being piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. In the mean time, Lokanath & Kunishima (2006) successfully determined the structure of *Tth* ICDH at 1.8 Å resolution. It should be instructive to mention an overview of typical ICDH structures, and what are still obscure and open to discussion from the view of structural biology and enzymology of ICDHs picking up some representatives.

### **4.1** *Escherichia coli* **ICDH**

The ICDH are usually dimeric proteins with two identical subunits of molecular mass of 40 - 50 kDa per subunit (Chen & Gadal, 1990). In *E. coli*, ICDH is a homodimeric enzyme and its inactivation mechanism by phosphorylation has been reported in detail with regards to the crystal structure (Hurley et al., 1990). The crystal structure of ICDH from *E. coli* (*Ec* ICDH) shows that the substrate binding pockets and catalytic sites of the dimeric enzymes are formed from side chains of residues donated asymmetrically both subunits (Hurley et al., 1989). The tertiary structure of *Ec* ICDH is depicted in Fig. 8. The enzyme is composed of 13 α-helices and 14 β-strands. It contains three domains consisting of a large domain, a small domain, and a clasp domain. This manner is common to ICDH from *Bacillus subtilis* (*Bs* ICDH) and *Tth* ICDH. This enzyme has an active site in a cleft between the large and small domains (Hurley et al., 1994; Stoddard et al., 1993). The reaction mechanism of ICDH has been extensively studied in *E. coli* ICDH. A conformational change occurs from an open to closed form upon the binding of NADP+ and the substrate. In the proposed mechanism, a proton is removed from the α-hydroxyl group of isocitrate, and then, a hydride ion is transferred in a stereospecific way from the α-carbon atom of the substrate to C-4 of the nicotinamide ring of NADP+, oxidizing isocitrate to oxalosuccinate. In the following step, the β-carboxylate group of oxalosuccinate is removed as CO2, and is replaced by a proton in a stereospecific way to form 2-oxoglutarate. During both transitions the negative charge on the hydroxyl oxygen atom of isocitrate is stabilized by a magnesium ion. There are still controversies as to the mechanisms of the initial proton abstraction and the final proton donation.

Crystallization, Structure and Functional Robustness of Isocitrate Dehydrogenases 323

presented with a racemic mixture of isocitrate in the presence of Mg2+, only D-isomer is seen in the active site in the crystal structure. They indicate the importance of the -OH group of L-isocitrate association with arginine residue at position 119 in the metal-free enzyme, in contrast to the -OH group of D-isocitrate association with the metal and with two aspartate

Arg 129 Asp 311

Mg2+

Tyr 160

Recently, the crystal structure of *Bs* ICDH has been reported and discussed on the difference between *Ec* ICDH. Both have very similar 3-D structures not only because *Bs* ICDH is 67% identical to *Ec* ICDH but also that both are 100% identical in the primary sequence around the phosphorylation site (Singh et al., 2001). The tertiary structure of *Bs* ICDH monomer A is illustrated in Fig. 10, and the *Bs* ICDH dimer is shown in Fig. 11. Each subunit is composed of 15 α-helices and 13 β-strands. Although *Bs* ICDH is a homodimer, interestingly, the crystal structures of the individual monomers are not identical. In the dimerization*,* socalled clasp domain is formed where constituent of two β-strands and connecting α-helix of each subunit interlock forming a hydrophobic core. The manner of intersubunit interactions in *Bs* ICDH dimer is reported the same for the most part with *Ec* ICDH. There are several reported differences between *Bs* ICDH and *Ec* ICDH, suggesting the robustness of the

Asp 283'

Arg 153

Asp 307

Lys 230'

residues at position 283' and 307.

Ser 113

Fig. 9. Overview of the ICDH substrate binding site.

enzyme in preserving the principle function.

**4.2** *Bacillus subtilis* **ICDH** 

Arg 119

Isocitric acid

The crystal structure of *Ec* ICDH determined with its substrates revealed that the malate moiety of isocitrate, namely 1-carboxyl and 3-carboxyl groups and 2-hydroxyl group, is recognized by three arginine, three aspartate, one lysine, and one tyrosine residues of ICDH, which are the residues conserved between the enzymes (Hurley et al., 1990). As shown in Fig. 9, in the substrate binding site, the α-carboxyl group of isocitrate is bound to three arginine residues, Arg-119, Arg-129, and Arg-153. The β-carboxylate group is bound to Arg-153 and Tyr-160. The α-position of isocitrate forms hydrogen bonds with the Lys-230', and Asp-283' residues of the other subunit of the dimer (Doyle et al., 2001; Mesecar et al., 1997).

Fig. 8. Diagram of *Ec* ICDH monomer with isocitric acid. (PDB ID: 1P8F). The large domain containing the N and C termini, the small domain, and the clasp domain are indicated. Atomic coordinates were obtained from the RCSB Protein Data Bank (www.rcsb.org/pdb/home/home.do) and imaged using PyMol (The PyMOL Molecular Graphics System, version 0.99, DeLano scientific, LLC).

Mesecar and Koshland Jr. have made precisely explanation as to the mechanism for stereospecificity, that is, to distinguish between the two enantiomers on the basis revealed by electron density maps of the crystal structures of ICDH with L- and D-isomer isocitrate (Mesecar & Koshland Jr., 2000). In the metal-free crystal of ICDH with a racemic mixture of isocitrate, only the L-isomer (2S,3R) is seen bound to the enzyme. When enzyme crystals are presented with a racemic mixture of isocitrate in the presence of Mg2+, only D-isomer is seen in the active site in the crystal structure. They indicate the importance of the -OH group of L-isocitrate association with arginine residue at position 119 in the metal-free enzyme, in contrast to the -OH group of D-isocitrate association with the metal and with two aspartate residues at position 283' and 307.

Fig. 9. Overview of the ICDH substrate binding site.
