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

322 Current Trends in X-Ray Crystallography

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).

Large domain

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.

(www.rcsb.org/pdb/home/home.do) and imaged using PyMol (The PyMOL Molecular

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

Atomic coordinates were obtained from the RCSB Protein Data Bank

C

Graphics System, version 0.99, DeLano scientific, LLC).

Small domain

N

Isocitric acid

Clasp domain

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 enzyme in preserving the principle function.

Crystallization, Structure and Functional Robustness of Isocitrate Dehydrogenases 325

Fig. 12. Amino acid sequence alignment of *Tth* ICDH, *Ec* ICDH, and *Bs* ICDH. The shaded and boxed sequences represent those identical to *Tth* ICDH sequence, and boxed ones

represent those in similar to *Tth* ICDH sequence.

Fig. 10. Cα trace of *Bs* ICDH monomer (monomer A) with citric acid. (PDB ID: 1HQS). Overall folding manner of the monomer resembles that of *Ec* ICDH, but has several differences in the secondary structures.

Fig. 11. Diagram of *Bs* ICDH dimer. Each of monomer A, and monomer B having slightly different conformation are related by dimerization mediated through expansive portion of small and clasp domains. (PDB ID: 1HQS).

Citric acid

N

C

C N

differences in the secondary structures.

C

N

small and clasp domains. (PDB ID: 1HQS).

Fig. 10. Cα trace of *Bs* ICDH monomer (monomer A) with citric acid. (PDB ID: 1HQS). Overall folding manner of the monomer resembles that of *Ec* ICDH, but has several

Subunit A Subunit B

Clasp region Fig. 11. Diagram of *Bs* ICDH dimer. Each of monomer A, and monomer B having slightly different conformation are related by dimerization mediated through expansive portion of


Fig. 12. Amino acid sequence alignment of *Tth* ICDH, *Ec* ICDH, and *Bs* ICDH. The shaded and boxed sequences represent those identical to *Tth* ICDH sequence, and boxed ones represent those in similar to *Tth* ICDH sequence.

Crystallization, Structure and Functional Robustness of Isocitrate Dehydrogenases 327

Fig. 14. Diagram of *Tth* ICDH dimer. (A) Monomer A is shown in blue with its 141 extra residues at the C terminus region in red. Monomer B is shown in pale-green. (B) in the view,

Although more than 40 structures including many mutants of homo-dimeric ICDH are available from Protein Data Bank (RCSB PDB), there are few structures with as clearly drawn electron density as the nicotinamide ribose moiety for example. It is understood that there lies the difficulty due to disorder, hence, further structural information is still requisite to elucidate the catalytic mechanism. Observation by AFM of the surface of the crystal form II of *Tth* ICDH suggested that the crystals consisted of huge ellipsoidal bodies of a homocomplex of ICDH, of which long axis' diameter was 18.6 nm and short one was 10.9 nm. The

*Tth* ICDH has been rotated ~180° around the axis indicated in (A). (PDB ID: 2D1C).

**A**

**B**

**5. Conclusion** 

#### **4.3** *Thermus thermophilus* **ICDH**

Between *T. thermophilus* and *E. coli* ICDHs, the residues of 37% are identical and those of 51% show similarity. The typical difference in the primary structure between the two enzymes is the presence of 141 extra residues at the C terminus in *Tth* ICDH. The region may contribute to the folding of the enzyme and the acquired thermostability of the enzyme (Miyazaki et al., 1992). Between *Tth* ICDH and *Bs* ICDH, the residues of 35% are identical and those of 50% have similarity. The primary sequence alignment of *Tth* ICDH, *Ec* ICDH, and *Bs* ICDH is shown in Fig. 12. Enzymes from thermophiles are often highly homologous to the mesophilic counterparts and the catalytic mechanisms are usually identical. The thermophilic enzymes have to be stable enough to withstand denaturation at elevated temperature where the thermophile optimally grows and possess simultaneously the flexibility required for enzymatic activity. Comparison among these enzymes of functions and structural similarities suggest functional robustness. The crystal structure of *Tth* ICDH is shown in Fig. 13. The overall core structure resembles those of *Ec* ICDH and *Bs* ICDH. As shown in Fig. 14, it can be seen that the string of the extra residues constitutes clasp domain, and it appears playing a role in the formation of a stable dimer.

Fig. 13. Cα trace of *Tth* ICDH monomer with citric acid. (PDB ID: 2D1C). Although there is a string of the extra sequence at the C terminus region, overall folding manner is quit similar to *Ec* ICDH and *Bs* ICDH. It may the reflection of functional robustness of the protein.

Although the preliminary X-ray study and crystallization of *Tth* ICDH were already reported by Ohzeki et al. (1995), and quite recently the crystal structure was determined by Lakonath & Kunishima (2006), we have described the other two curious crystal forms (crystal form I, and II) obtained for *Tth* ICDH under different crystallization conditions. Observation on the surface of one of the crystal forms (form II) by AFM motivated extended analysis on the possibility of *Tth* ICDH molecule taking the form of a supramolecular architecture under the condition and crystallization state. Study in this line leads to the important subject which concerns polymorphism of protein crystals, and has focused on the importance of spontaneous hierarchical construction with supramolecular assemblies as a block towards nano-composites.

Fig. 14. Diagram of *Tth* ICDH dimer. (A) Monomer A is shown in blue with its 141 extra residues at the C terminus region in red. Monomer B is shown in pale-green. (B) in the view, *Tth* ICDH has been rotated ~180° around the axis indicated in (A). (PDB ID: 2D1C).
