# Glucose Isomerase: Functions, Structures, and Applications
## 3 Structure  
### Overall Structure  
The first crystal structure of GI was reported from _Streptomyces rubiginos_ at a 4 A resolution in 2001 [21]. To date, more than 120 crystal structures of GI/XIs from various species, such as _S. rubiginosus_, _Arthobacter_ sp. NRRL B3728, _Piromyces_ sp. E2, _Actinoplanes Missouriensis_, _Streptomyces olivochromogenes_, _Streptomyces_ s. F-1, _Streptomyces_ sp. SK, _Bacteroides thetaiotaomicron_ VPI-5482, _Geobacillus stearothermophilus_, _Paenibacillus_ sp. R4, _Streptomyces albus_, _S. murinus_, _Thermaaneroducerium thermosulfurigenes_, _Thermotoga neapolitana_, _Thermus calophilus_, and _Thermus thermophilus_ HB8, have been elucidated and deposited in the Protein Data Bank (PDB, www.rcsb.org). These include more than 100 crystal structures of the GI from _S. rubiginosus_; however, many of these structures have been solved and used only as model samples for the development of various X-ray technologies or studying radiation damage, because the GI from _S. rubiginosus_ exhibits high-quality diffraction intensity [22; 23; 24; 25; 26; 27; 28; 29].  
Structurally, GI consists of a TIM barrel-like domain and an extended \(\alpha\)-helix domain (Figure 3A,B) which are assembled into a functional tetramer (Figure 3C) [21]. The substrate-binding pocket is formed by two protomers and the GI active site is located on the TIM-barrel fold (Figure 3C). The GI exists as a tetramer with 222 crystallographic symmetry [30] and includes four active sites for substrate isomerization.  
Figure 3: Crystal structure of the GI from _Streptomyces rubiginos_ (SruGI). (**a**) GI protomer consists of a TIM-barrel domain and an extended \(\alpha\)-helical domain. (**b**) The extended \(\alpha\)-helical domain interacts with the TIM barrel domain from neighboring GI molecule. (**c**) Tetrameric GI shows 222 symmetry.  
Figure 2: Isomerization mechanism of GI. (**a**) Three major steps involved in the configuration change from D-glucose to D-fructose catalyzed by GI. (**b**) Hydride shift mechanism of GI.  
The typical GI active site contains two sites, M1 and M2, for metal binding. M1 and M2 are referred to as the structural metal and catalytic metal sites, respectively [31], as the metals bound at the M1 and M2 sites are involved in the substrate binding and isomerization mechanism, respectively. In _S. rubiginosus_, the metal at the M1 site is coordinated by Glu181, Glu217, Asp245, and Asp287, and the metal at the M2 site binds the protein via Glu217, His220, Asp255, and Asp257. These metal-bound amino acids are conserved across the GI family [32; 33] (Figure 4).  
Most of the crystal structures of GI are bound to the metal ions Mg\({}^{2+}\), Mn\({}^{2+}\), or Co\({}^{2+}\), which are involved in the isomerase activity [1]. However, some GI crystal structures are known to contain biologically less related metal ions in their active sites [34; 35]. For example, in the crystal structures of GIs from _S. rubiginosus_ (PDB code: 4W4Q) [36], _Paenibacillus_ sp. _R4_ (PDB code: 6 INT) [34], and _Pironyces_ (6T8E) [35], Ca\({}^{2+}\) is located in the M1 and M2 sites of the active site, although enzyme activity in the presence of Ca\({}^{2+}\) has not been verified. In addition, in some crystal structures, the metal ion was placed in a position other than at the M1 and M2 sites [37]. Thus, metal ions in the crystal structures of GI require verification through biochemical experiments or X-ray analysis; additionally, precise analysis of electron density is required to confirm their position and involvement in GI substrate binding and activity.  
Figure 4: Amino acid sequence alignment of GIs from _Streptomyces rubiginosus_ (UniProt Accession: P24300), _Arthobacter_ sp. NRRL B3728 (P12070), _Pironyces_ sp. E2 (Q9 PSC9), _Actinoplanes missouriensis_ (P12851), and _Streptomyces olivochromogenes_ (P15587). Residues involved in metal binding at the M1 and M2 sites are indicated by red and blue triangles, respectively. Alignment was performed using Clustal Omega [38] and displayed using Espript [39].