Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.
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.
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3/VI, A-6020 Innsbruck, Austria.
Katrin
K
Watschinger
Julian E
JE
Fuchs
Vladimir
V
Yarov-Yarovoy
Markus A
MA
Keller
Georg
G
Golderer
Albin
A
Hermetter
Gabriele
G
Werner-Felmayer
Nicolas
N
Hulo
Ernst R
ER
Werner
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D016466:CHO Cells; D020134:Catalytic Domain; D003198:Computer Simulation; D016384:Consensus Sequence; D006224:Cricetinae; D006801:Humans; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D001708:Biopterins; D007501:Iron; D006899:Mixed Function Oxygenases; C020678:glyceryl-ether monooxygenase; C003402:sapropterin