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Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

Ischemia-reperfusion injury is a primarily non-allospecific event leading to the depletion of the essential nitric oxide synthase cofactor and potent antioxidant tetrahydrobiopterin. Suboptimal concentrations of tetrahydrobiopterin result in a reduced biosynthesis of nitric oxide leading to vascular endothelial dysfunction. Tetrahydrobiopterin supplementation has been shown to protect from this pathological state in a plethora of cardiovascular diseases including transplant-related ischemia-reperfusion injury. Even though still controversially discussed, there is increasing evidence emerging from both human as well as animal studies that tetrahydrobiopterin-mediated actions rely on its nitric oxide synthase cofactor activity rather than on its antioxidative properties. Herein, we review the current literature regarding the role of tetrahydrobiopterin in ischemia-reperfusion injury including our experience acquired in a murine pancreas transplantation model.

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O-alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzyme.

The tetrahydrobiopterin-dependent degradation of ether lipids by alkylglycerol monooxygenase (AGMO) produces fatty aldehydes, which are toxic to cells. Therefore, it is of great physiological importance that these harmful compounds are converted into their corresponding, less toxic fatty acids by fatty aldehyde dehydrogenase (FALDH). Dysfunction of this enzyme causes Sjögren-Larsson syndrome. This severe inherited disorder is accompanied by symptoms such as ichthyosis, mental retardation and spasticity. Surprisingly, fatty alcohols and not fatty aldehydes were found to accumulate in fibroblasts of Sjögren-Larsson syndrome patients, suggesting that there can be wide-ranging alterations in the lipid composition of patient cells. In particular, this has to be considered when searching for possible treatment options for patients suffering from Sjögren-Larsson syndrome. For example, inhibition of fatty aldehyde producing ether lipid degradation would have multiple implications on ether lipid- and fatty alcohol-mediated signalling pathways.

Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases – comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases – and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N5-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis. [ABSTRACT FROM AUTHOR]