**5. Conclusion**

326 Current Trends in X-Ray Crystallography

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,

C

Citric acid

and it appears playing a role in the formation of a stable dimer.

N

block towards nano-composites.

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

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

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

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HPLC gel filtration column chromatography of the protein co-existed with its form II crystals which had been incubated under the critical crystallization condition, thus supporting the line of the above assumption. The calculation using the preliminary X-ray diffraction data indicated that an asymmetric unit contains lots of oligomeric *Tth* ICDH in crystal form II. We have found huge supramolecular complex formation under the appropriate crystallization condition among the ICDH subfamilies, which concerns polymorphism of protein crystals.
