XyloseIsomerase

Xylose isomerase (6YBR) is a protein found in the bacterium Streptomyces rubiginosus that catalyzes the interconversion of D-sugars. It has been observed in many species of prokaryotes and is important in the conversion of various types of sugars, such as D-xylose, D-glucose, and D-fructose. Xylose isomerase has been the subject of many intensive research efforts and as a result, it serves as a useful model for studying structure-to-function relationships using biochemical and bio-engineering techniques. In addition, xylose isomerase has an important industrial use in the production of high-fructose corn syrup and ethanol (1).

Xylose isomerase is produced by the bacterium Streptomyces rubiginosus, which is found in soil. Slightly alkaline environments are optimal for isomerase activity, but overall activity is low in most species. For maximum activity, a divalent ion such as Mg2+ is required, as it plays an important role in holding the quaternary structure together. Crystals of xylose isomerase may be grown in situ on Mylar and Kapton microchips at room temperature using counter diffusion x-ray crystallization techniques (2). Xylose isomerase was specifically used as a model protein for the crystallization method utilizing these microchips, in hopes of developing a new low-cost method.

Xylose isomerase is a large globular protein with molecular weight 43227.37 Da and isoelectric point of 5.00 (3). Overall, it is made of four identical subunits that are linked together and group around a central active site. Xylose isomerase consists of two structural domains. The larger of the two domains consists of units arranged in a barrel-like configuration. The smaller domain forms a loop that branches away from the large domain and overlaps with the large domain of another subunit to form a dimer (4). The total sequence of xylose isomerase has a length of 388 residues; it has been studied enough to the point that there are no unknown/unmodeled regions.

The primary structure of xylose isomerase consists of 388 residues with many repeats of nonpolar amino acids such as alanine, leucine, and isoleucine that contribute to forming hydrophobic regions. The secondary structure consists of repeating sequences of alpha-helices and beta-pleated sheets. They are sequenced back to back so that the backbone can loop repeatedly to organize the helices and sheets (4). Because beta-pleated sheets have many hydrophobic side chains, this sequencing better allows the protein to fold to form a hydrophobic center when the subunits are connected. Figure 1 shows the backbone structure of xylose isomerase. Figure 1 is particularly useful for showing the large domain, where alpha-helices are distributed so that they surround the inner beta-pleated sheets around the active site (4). The tertiary structure organizes the helices and sheets so that the alpha-helices are gathered towards the outside and the beta-pleated sheets gather around the center. A sequence of alpha-helices also serves to form the loop that corresponds with the small domain. The quaternary structure is held together by dimerization of the small domain loop with the large domain, along with interactions with metal cations Mg2+ and Na+ (5). Figure 2 shows the xylose isomerase tetramer viewed along the y-axis (4).

The overall structure of xylose isomerase allows the active site in its center to bind to its substrates D-xylose and D-fructose. Several ligands and residues have been identified as serving important roles in the xylose isomerase active site. Mg2+ and Na+ are metal ions that contribute to active site function and structure. Mg2+ is a ligand that is integral for the binding of the substrate to the active site (5). Na+ contributes to subunit structure in the smaller domain. Two histidine residues (His-101 and His-271) were identified. His-101 is believed to be a catalytic base that mediates the reaction. His-271 is believed to be a ligand for the metal cations in the active site. Xylose isomerase from purified from E-coli to test this hypothesis. When the codons corresponding to these histidine residues are substituted via mutagenesis to form different amino acids, the resulting mutant xylose isomerases exhibited no activity (6). Glu-216, Lys-289, and His-53 are other notable residues that have functions in the active site. Glu-216 bridges the two metal Mg2+ cations in the active site. Lys-289 catalyzes a hydride shift in the isomerization of glucose, and His-53 catalyzes proton transfer of oxygen atoms in the same isomerization (6). Notably, His-53 also forms hydrogen bonds with the nearby Asp-56 residue, which activates it. All these factors contribute to xylose isomerase binding with its substrate sugar, and the result has many industrial applications. Xylulose created from the isomerization of xylose is present as a final product in high-fructose corn syrup (which is sweeter than sucrose); the xylulose can also be fermented by yeasts to produce ethanol (1).

Xylose isomerase is conserved in many prokaryotic species beyond Streptomyces rubiginosus. Similarities between different isomerases produced between organisms can be explored using PSI-BLAST and Dali methods. PSI-BLAST produces a measure of the similarity between the primary structures of two different proteins and returns an E-value. The closer the E-value is to zero, the closer the match. The Dali method compares and finds proteins with similar tertiary structures via a sum-of-pairs method. It compares intramolecular distances and returns a Z-score as a measure of similarity. Z-scores above 2.0 indicate very high similarity.

PSI-BLAST and Dali queries of xylose isomerase were performed and many copies of xylose isomerase from different species were analyzed. Because xylose isomerase is such a highly conserved protein among many different species of bacteria, all of the E-values corresponding to results from the query were 0.0, indicating an identical or near-identical primary structure (7). Similarly, the Dali query returned copies of xylose isomerase from different species with very high Z-scores (8). The xylose isomerase recovered from Streptomyces olivochromogenes (PDB ID: 1S5N) had an E-value of 0.0 and a Z-score of 60.5. A superimposed view of the structures between the two isomerases indicate that they are virtually identical. They are composed of the same backbone structure, with only minimal differences in intramolecular distances. The overall positioning of alpha-helices and beta-pleated sheets are the same, with the alpha-helices forming an outside layer around the beta-pleated sheets. The isomerase from Streptomyces olivochromogenes was noted to rely on separate ligands to promote activity, these being: Mn2+, Na+, OH-, and xylitol (6). Though the ligands for the two isomerases are different, a side-by-side comparison shows that their active sites are still in the same positions and have the same function.