Watschinger, Katrin Fuchs, Julian E. Yarov-Yarovoy, Vladimir Keller, Markus A. Golderer, Georg Hermetter, Albin Werner-Felmayer, Gabriele Hulo, Nicolas Werner, Ernst R. 2012 <jats:p>Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.</jats:p> application/pdf https://sonar.ch/global/documents/190067 eng info:eu-repo/semantics/altIdentifier/doi/10.1042/BJ20111509 info:eu-repo/semantics/altIdentifier/issn/0264-6021 info:eu-repo/semantics/openAccess Biochemical Journal. - Portland Press Ltd.. - 2012, vol. 443, no. 1, p. 279-286 Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase http://purl.org/coar/resource_type/c_6501