Choline Oxidase

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Introduction

Choline oxidase (choline-oxygen 1-oxidoreductase) catalyzes the four-electron, two-step, flavin-mediated oxidation of choline to glycine betaine. In the first step, choline is oxidized to betaine aldehyde, and then molecular oxygen is reduced to hydrogen peroxide. In the second step, the betaine aldehyde is hydrated to yielding gem-diol-choline, which is oxidized to glycine betaine, and oxygen is reduced to hydrogen peroxide. The enzyme was first isolated from rat liver in 1948 when studying the effects of nitrogen mustards on enzymes and tissue metabolism. Later, Ikuta et al. first identified purified choline oxidase from Arthrobacter globiformis in vitroin 1977. Glycine betaine, the product of choline oxidase, is one of a limited number of biocompatible solutes used by cells to compensate for high osmotic pressure. The accumulation of large amounts of glycine betaine in the cytoplasm is essential for microorganisms because they can control the intracellular water content in the cells. Failure to accumulate glycine betaine in a high-salt environment will result in dehydration, osmotic shock, and plasmolysis. Many human pathogens including E. coli O157:H7, Staphylococcus spp., Enterococcus faecalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae,accumulate glycine betaine to compensate for the high osmolality encountered at the site of human infection.

Glycine betaine is an important osmoprotectant in human kidney, and is also used as a methyl donor in the biosynthesis of methionine. In humans, choline can play a role in different pathways, including biosynthesis of lipoprotein and phospholipid, regulation of gene expression. Human choline dehydrogenase is closely related to a variety of pathologies, including cardiovascular disease, breast cancer, male infertility, homocysteinuria, and metabolic syndrome, etc.

Figure 1. The two-step, flavin-mediated oxidation of choline to glycine betaine catalyzed by choline oxidase (Gadda, G. 2020)

Enzyme structure

The results of size exclusion chromatography and nondenaturing polyacrylamide gel electrophoresis confirmed that choline oxidase in solution exists as a concentration-independent dimer with a molecular weight of 120 kDa. A variety of crystal structures, such as the crystal structure of wild-type enzyme and with glycine betaine bound and with DMSO-bound in the active site cavity, the crystal structure of the active site mutants S101A in complex with acetate, and the crystal structure of V464A devoid of ligands are all confirmed the dimer state of the enzyme. The dimer interface consists of two sets of six identical inter-subunit contacts between residues with the opposite charge at the interface edges (Figure 2).

Figure 2. Three-dimensional structure of choline oxidase refined to a resolution of 1.86Å (Protein Data Bank 2jbv) (Gadda, G. 2020)

The central part of the dimer interface includes a set of non-polar residues that act as a gate to allow substrate entry and products to enter. The folding mode of each subunit of choline oxidase is similar to that of other glucose-methanol-choline oxidoreductases, with a PHBH folding. The enzyme has a FAD-binding domain (consisting of residues 1-159, 201-311, and 464-527), and a substrate-binding domain (consisting of residues 160-200 and 312-463). The flavin is tightly embedded in the protein and is covalently linked to the H99Nε2 atom through the C8 methyl. In the substrate binding domain, the bottom of the active site is formed by distorted six-stranded antiparallel β-sheets, and on the other side are three α-helices protruding into the bulk solvent. The active site cavity contains residues from both domains, E312, W331, H351 and F357 in the substrate-binding domain, and W61, S101, W331, V464, H466 and N510 in the FAD-binding domain (Figure 3).

Figure 3. Active site of wild-type choline oxidase with the reaction product glycine betaine Bound (Gadda, G. 2020)

Substrate binding

The active site cavity of choline oxidase is solvent-excluded and is adjacent to the reface of the FAD. The volume of the cavity is ~125 ų, which is enough to accommodate a choline molecule (~95 ų) and a H₂O molecule (~30 ų). The part of the cavity away from flavin is surrounded by hydrophobic residues (ie, W61, W331, F357, and V464). The carboxylic side chain of the only negatively charged residue E312 contributes ~15 kJ/mol to the binding of the substrate through the ionic interaction with the trimethylammonium headgroup of choline. X-ray crystallography and mutagenesis studies have shown that the three polar residues border the active site cavity near the isoalloxazine portion of the FAD, namely H351, H466 and N510, have their side chain contributes less to the binding of the substrate by either hydrogen bond interactions with the hydroxyl of choline or steric effects.

Figure 4. Entrance to the active site of choline oxidase (Gadda, G. 2020)