Oxalate oxidase (PDB ID: 2ET1) from Hordeum vulgare

Created by: Jenny Oh

            Oxalate oxidase (PDB ID: 2ET1) from Hordeum vulgare is the enzyme that catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide (oxalate + O₂ + 2H⁺ ↔ 2CO₂ + H₂O₂). Oxalate oxidase is widespread and found in many different organisms, such as bacteria, fungi and various plant tissues. There has been a suggestion of its role in plant signaling and defense, and also there has been the recognition that the oxalate oxidase enzyme is identical to an important marker of grain development during germination of wheat called germin (1). Plant cells express oxalate oxidase as an important countermeasure to fungal invasion by eliminating the oxalic acid and producing hydrogen peroxide, which can serve both as a fungicidal agent and as a signal for plant defenses and development (2). In addition to these biological roles, oxalate oxidase has significant bioanalytical applications and is used in the clinical determination of oxalate in biological fluids. Oxalate oxidase also has potential applications in treatment of calcium oxalate kidney stones and prevention of formation of calcium oxalate deposits in paper manufacture (3).

Oxalate oxidase was fully crystallized using hanging-drop vapor diffusion method at 18°C. For this, 1µl of protein (10-15 mg/ml) was mixed with 1-µl reservoir drops and 1-ml reservoirs. Crystals of oxalate oxidase grew with PEG 4000 or 8000 in the pH range 4.6-8.5 and depending on the particular combination of conditions, different types of crystals were produced (4). Native crystals that were grown from 2.3 M (NH₄)₂SO₄ and 5% 2-propanol at 298K and in pH 6 were rhombohedral with one subunit in the asymmetric unit (1). For structural data, x-ray diffraction was used (5). The subsequent crystallographic studies revealed the structure of the hexamer. The native crystals rhombohedral R32 was a = 96.3Å, c = 108.1Å (hexagonal setting). The resolution of the protein was 1.6Å and the R-free value was 0.206 (1). Spectroscopic studies and crystallographic studies confirmed that oxalate oxidase requires a mononuclear manganese for catalysis. The addition of oxalate to the manganese resulted in the production of carboxylic free radicals (1).

According to Expasy, the isoelectric point of oxalate oxidase is 5.52 and its molecular weight is 21,217.22 Da (6). Oxalate oxidase has a total of 201 residues in one chain and is composed of both hydrophobic and polar amino acids. There is only one unique polypeptide chain A, and the global stoichiometry of the enzyme is homo-6-mer, A6 (5). The assembly of multiple identical subunits provides very efficient ways of constructing large structure and allows more complex processes (7). The identical subunits may also allow multiple catalysis to occur at once. Oxalate oxidase contains 12 beta strands with 58 residues (28% of the structure) and 6 helices with 33 residues (16% of the structure). 3 out of 6 helices are 3/10-helix with 9 residues. The random coils are present in the structure but to an uncertain degree.

There are two ligands, glyoxylic acid and manganese (II) ion, present in the crystal structure of oxalate oxidase. Glyoxylic acid serves no biological function and is used merely to induce crystallization (1). Manganese (II) ion organizes the substrates, oxalate and dioxygen, and transiently reduces dioxygen. Three specific active site residues (Asn-75, Asn-85 and Gln-139) also serve important roles in correctly orientating substrates and reaction intermediates for catalysis. All three residues are poised to hold the carboxylic substrate and dioxygen in the correct orientation with respect to the manganese for catalysis. Asn-75 and Asn-85 can also stabilize catalytic intermediates, while Asn-85 and Gln-139 ensure that manganese, carboxylate, and dioxygen radical are planar to direct the formation of the percarbonate product (1).

The manganese ion at the active center of oxalate oxidase is bound by three histidines (His-88, His-90, His-137), one glutamate (Glu-95) and two waters (Figure 1) (1). The crystallographic results has shown that oxalate binds to the manganese ion using one carboxylate oxygen in-plane and displaces one of the coordinating water molecules from the active site (Figure 2) (1). Another carboxylate oxygen also forms a hydrogen bond with the remaining manganese-bound water molecule. Asn-75 changes its conformer to form a hydrogen bond with the hydroxyl hydrogen, and the conformational flexibility of the Asn-75 side chain suggests a possible dynamic role for its mobility in assisting movement of substrates and products (1). The network of hydrogen bonds anchoring glycolate in the oxalate oxidase active site also includes the side chain of Gln-139. Gln-139 forms hydrogen bonds with both Asn-85 and the manganese-bound water molecule (Figure 2) (1).

The Dali server compares tertiary structures of proteins and calculates the difference in intramolecular distances via a sum-of-pairs method. A Z score is considered significant if it is greater than 2. According to the Dali server, the protein with the most structural similarity is glycinin A3B4 subunit (PBD ID: 2D5F), which is the major seed storage protein of soybean with a Z value of 16.2 (8). Glycinin A3B4 subunit is also a hexamer and each subunit is composed of an acidic and basic chain derived from a single precursor and linked by a disulfide bond. Because it has 2 chains, glycinin A3B4 subunit has twice the number of both helices and beta sheets strands compared to oxalate oxidase. The hexamer formation of glycinin A3B4 subunit is due to thermal stabilization, which may have been partly affected by the change in hydrophobic residues on the molecular surface brought about by the cleavage. These exposed hydrophobic residues form hydrophobic interactions with the other trimer and may have led to the formation of hexamer (9). Similar to oxalate oxidase, Asn-135 plays an important role in glycinin A3B4 subunit. This amino acid maintains a globular structure and because of its polarity, its side chain can form hydrogen bonds with the peptide backbone, which impart stability (9). Unlike oxalate oxidase, glycinin A3B4 subunit has 2 ligands, magnesium (II) ion and carbonate ion, and may more “globally conserved” residues (9). The highly conserved residues,Phe-208, Leu-214, Leu-227and Val-466, participate in alpha helix formation, and 4 glycinin residues, Gly-28, Gly-74, Gly-349 and Gly-397, found in beta turns are key amino acids in maintaining the globular structure of the protein (9). Glycinin A3B4 subunit and oxalate oxidase have similar tertiary and quaternary structures, but Glycinin A3B4 subunit did not appear as a homology according to PSI-BLAST.

PSI-BLAST finds proteins with similar primary structures to a protein query, where an E value less than 0.05 indicates that there is a significant similarity between two protein structures. According to PSI-BLAST, the protein with the most similarity in primary structure is SLL1358 protein (PDB ID: 2VQA) with an E value of 0.003 (10). SLL1358 protein is the crystal structure of manganese (II)-cupin A protein (MncA), present in the periplasm of the cyanobacterium Synechocystis PCC 6803 (11). Unlike oxalate oxidase, SLL1358 protein is homo-3-mer with three chains of 361 amino acids each, so there is 26.28% primary structure similarity between the two proteins (10). There are also two ligands, manganese (II) ion and acetate ion (11). The Dali server has generated a Z score of 15 for SLL1358 protein  which indicates that there is a significant similarity in the tertiary structure, but there are no functional homologies between SLL1358 subunit and oxalate oxidase.

Oxalate oxidase has been used to produce transgenic crops with improved tolerance to fungal pathogens, such as Sclerotinia sclerotiorum that use oxalic acid as a toxin (4). There has been a suggestion that transgenic crops with elevated oxalate oxidase expression levels may not exhibit higher enzyme activities nor fulfill their potential for increased fungal resistance if manganese ions were limiting in seed and soil. As such, the severity of take-all infection of wheat caused by the fungus Gaeumannomyces graminis could be related to the manganese ion content of seed and may be associated with oxalate oxidase activity (12). All members of cupin superfamily, including oxalate oxidase, are capable of binding specific metal ions that have distinct catalytic and structural roles. However, not all members bind to manganese ion; some of the germin-like proteins are thought to bind other metal ions, such as iron ion and possibly zinc ion (12). Therefore, further studies on the significance of binding to different metals and their distinct catalytic and structural roles would provide a better understanding on germin-like proteins.