Journal
BIOCHEMICAL JOURNAL
Volume 443, Issue -, Pages 279-286Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20111509
Keywords
alkylglycerol mono-oxygenase; fatty acid hydroxylase motif; phenylalanine hydroxylase; tetrahydrobiopterin; transmembrane domain
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Funding
- Austrian Science Fund (FWF) [P22406-B18]
- Swiss National Science Foundation (SNSF) [315230B-131083]
- Swiss State Secretariat for Education and Research (SER)
- Austrian Science Fund (FWF) [P22406] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [P 22406] Funding Source: researchfish
- Swiss National Science Foundation (SNF) [315230B_131083] Funding Source: Swiss National Science Foundation (SNF)
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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 monooxygenase, 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 Glu(137) 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.
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