Eugenol_Oxidase

Eugenol Oxidase (PDB ID: 5FXP), a flavoprotein oxidase from Rhodococcus jostii strain RHA1, is currently being studied to discover its full range of substrate selectivity and structural conformations (1, 2). Eugenol Oxidase is one of many flavoprotein oxidases, which are classified by the presence of a flavin moiety (2). The flavin moiety present in Eugenol Oxidase is flavin adenine dinucleotide (FAD), which serves as a redox cofactor critical to enzyme function, and is necessary to stabilize the dimeric form of Eugenol Oxidase (2). The biological and chemical functions of Eugenol Oxidase are of particular interest to scientists for synthetic applications such as oxidative procedures, because as a robust biocatalyst, its biotransformations result in high degrees of chemoselectivity, regioselectivity, and enantioselectivity for a wide range of organic solvents and temperatures (2).

The three-dimensional structure of Eugenol Oxidase was determined using the sitting-drop vapor diffusion technique, followed by X-ray diffraction, MOLREP molecular replacement software, then COOT and REFMAC5 model building and refinement software (2). Mass spectrometry experiments determined that 63 ± 2% of Eugenol Oxidase exists in the holo form, whereas 37 ± 2% of Eugenol Oxidase exists in the apo form, meaning that the FAD cofactor is absent (3). The most common, or holo, form of Eugenol Oxidase consists of two monomers that each have an approximate molecular weight of 59000 daltons and together form an asymmetric homodimer (2, 3). The primary structure of each monomer consists of 1,052 amino acids, of which approximately 15.3% form 25 β-sheets, 23.1% form 20 α-helices, and 61.6% form 41 random coils (4). The holo form of Eugenol Oxidase has a net molecular weight of 117343.31 daltons and an isoelectric point of 4.87 (5). The ExPASy server uses the primary structure of each monomer in Eugenol Oxidase to calculate these figures (5). The molecular weight of Eugenol Oxidase indicates that it is a relatively medium-sized on the scale of commonly known biological proteins, having a slightly lower molecular weight than that of Immunoglobulin (149900 Mr) found in horses (6). The isoelectric point of Eugenol Oxidase also represents the pH at which the flavoprotein is least soluble, as well as the net negative charge that Eugenol Oxidase is likely to hold at physiological pH (6).

The interface of the asymmetric homodimer in Eugenol Oxidase has a relatively large area of approximately 3500 Å2 , which represents 16.5% of each monomer’s surface area (2). Both monomers have a unique substrate binding cavity, which remain in close contact with a funnel-shaped invagination on the surface of the flavoprotein (2). The funnel-shaped invagination is created by the asymmetric homodimer interface and is believed to interact with the substrate-binding cavities of each monomer to form a passageway for substrate access to the inner, round-shaped active site of Eugenol Oxidase (2). The inner active site of Eugenol Oxidase is entirely solvent-inaccessible and is located directly beside the FAD cofactor and another prominent ligand, 4-hydroxy-3-methoxybenzaldehyde (2). The location of three residues, Gln-425, Leu-381 and Met-282, near the surface of the funnel-shaped invagination are responsible for narrowing the substrate-binding cavity of each monomer, which limit Eugenol Oxidase’s substrates to smaller, hydrophilic molecules rather than those with hydrophobic substituents and long alkyl chains (2). Small, specifically phenolic, molecules such as eugenol, vanillyl alcohol, and indan-5-ol with a rate of catalytic conversion into product of Kcat = 0.26–12 s-1 have been found as the most effective substrates for Eugenol Oxidase (2).

FAD and 4-hydroxy-3-methoxybenzaldehyde are two of Eugenol Oxidase’s critical ligands that affect substrate binding activity in the active site (2). FAD stabilizes the homodimeric form of Eugenol Oxidase because of a critical covalent bond with His-390, while it also uses its pyrimidine ring and the Cα of its aliphatic chain to bind with the phenol moiety of each substrate at a distance of 3.2 Å (2). The phenol moiety of each substrate is simultaneously stabilized in the active site by the ligand, 4-hydroxy-3-methoxybenzaldehyde, which forms a hydrogen bond with Tyr-91 and Tyr-471 (2). Arg-472 is another residue in close contact with 4-hydroxy-3-methoxybenzaldehyde at a distance of approximately 3.5 Å for further stabilization of a bound substrate’s functional groups through weak, electrostatic interactions with the guanidinium group on Arg-472 (2). The location of Tyr-91, Tyr-471, and Arg-472 near 4-hydroxy-3-methoxybenzaldehyde form an anion-binding site within the active site of Eugenol Oxidase, which also creates increased electron density around the FAD ligands of each monomer (2). The third ligand that is essential to Eugenol Oxidase biocatalytic activity is glycerol, which increases the stability of the homodimer during oxidation of bound substrates (7).

The asymmetric homodimeric structure and broad polarity of Eugenol Oxidase are directly related to its biological function as a flavoprotein oxidase. Once a substrate is bound to the active site it immediately goes through two to four electron oxidations in the presence of FAD (2). Reduced FAD is then reoxidized by molecular oxygen, which is able to easily travel through the narrow funnel-shaped passageway due to its small size (2). Eugenol Oxidase is also tolerant to a wide range of organic solvents, pH values, and temperatures, which is attributed to the inner location of the active site beneath the protein surface (2). Eugenol Oxidase also prefers phenolic substrates that contain ortho-hydroxyl-methoxy substituents, such as eugenol, over para-hydroxyl-methoxy substituents, such as 4-(methoxymethyl)-phenol, because of the close proximity of FAD and 4-hydroxy-3-methoxybenzaldehyde in the active site (2). The phenol moiety of the substrate aligns with FAD and 4-hydroxy-3-methoxybenzaldehyde simultaneously within a small region of the active site, which causes increased intermolecular interactions between both ligands and a substrate that has hydroxyl and methoxy substituents in the ortho-position rather than the para-position (2). A substrate with para-hydroxyl-methoxy substituents has limited intermolecular interactions with both ligands, because one of the substituents would be too far removed in space to be transformed (2).

An important comparison structure to Eugenol Oxidase is Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant (PDB ID: 1W1L) from Penicillium simplicissimum (1). Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant is a homooctamer flavoprotein with each subunit possessing an FAD cofactor and a molecular weight of 64000 daltons (8). Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant contains a characteristic His-422 covalent bond with FAD near the active site, similar to the His-390 covalent bond found in the active site of Eugenol Oxidase with FAD (2). The Z-score for Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant is 52.7, which was calculated using the Dali Server (9). The Dali Server calculates the difference in intramolecular distance between tertiary structures of proteins using the sums-of-pairs method (9). A Z-score greater than 2 is considered significant, indicating that Eugenol Oxidase and Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant have very similar folds (9). The E-score for Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant is 2.0×10-170, which was calculated using PSI-BLAST (10). PSI-BLAST searches PDB for primary structures that best match a query primary structure based on the number of gaps, or number of absent amino acids in the query protein compared to the query protein (10). An E-score below 0.5 is considered significant, indicating that Eugenol Oxidase and Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant have very similar primary structures (10). Previous studies have confirmed these figures by showing that Eugenol Oxidase and Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant share approximately 45% sequence identity (2).

The major difference between Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant and Eugenol Oxidase is the structure of the active site, which greatly alters substrate selectivity (2). In Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant, Phe-424 is found to replace Gly-392 from the active site of Eugenol Oxidase, which interacts with substrates that contain two methoxy groups (2). The bulkier Phe-424 of Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant most likely induces steric hindrance in the active site, causing a reversed binding orientation of substrates with methoxy groups by 180 degrees (2). Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant also contains a dimer-dimer interacting loop critical for octamer formation, which is not present in Eugenol Oxidase (2). Three particular residues found in the active site of Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant – Thr-457, Trp-413, and Leu-316 – are all replaced by three residues in Eugenol Oxidase that are responsible for the narrow shape of the active site cavity – Gln-425, Leu-381, and Met-282. The three residues found in the active site of Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant allow for a wider catalytic cavity that can accept substrates with aliphatic chains up to seven hydrocarbons long (2, 11). Previous studies have also found that Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant strictly requires para-hydroxyl substituents to the aliphatic chains of substrates for effective binding and biocatalysis, unlike Eugenol Oxidase (11).

Eugenol Oxidase has been shown to function in a wide range of experimental conditions, which makes it a flavoprotein oxidase of significant interest in the current field of biocatalytic redox reactions. It has also been demonstrated that the biological function of Eugenol Oxidase is a direct result of its unique asymmetric homodimeric structure with the location of the active site inside the protein and obstructed from access to the surface. Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant offers a highly similar sequence identity to Eugenol Oxidase, but the substrate specificity varies greatly between each flavoprotein oxidase because of alterations in critical residues of the active sites. The homooctamer structure of Vanillyl-Alcohol Oxidase: Phe454Tyr Mutant is also vastly different from the overall three-dimensional structure of Eugenol Oxidase due to the lack of a single dimer-dimer loop in the latter protein. Further research is being conducted into the full range of substrates and structural conformations specific to Eugenol Oxidase, which will present the means for creating a tailored biocatalyst for highly selective and large-scale oxidative biotransformations (2).