Abstract
Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys(127) in governing access to the diiron cluster.
Highlights
Myo-Inositol is a cyclitol that plays a crucial role in all eukaryotic cells by serving as a backbone for the most important second messengers: inositol phosphates and their lipid derivatives, phosphoinositides
The Hs-myo-inositol oxygenase (MIOX) has an overall topology that consists of eight ␣-helices and a small two-stranded antiparallel -sheet that act as a topological latch (Fig. 1)
In the vicinity of the active site, there are significant differences relating to the lack of the 37 N-terminal amino acids in the Hs-MIOX construct used here for crystallization
Summary
Cloning and Protein Production—For crystallization, DNA encoding residues 38 –285 of the human MIOX gene (gi:49522836) was cloned by ligation-independent cloning into a pET-28-based expression vector incorporating an N-terminal His tag fusion (pNIC-Bsa; gi:EF198016). MIOX (15 mg/ml in 20 mM Hepes, pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 5 mM myo-inositol, 1 mM L-cysteine, and 1 mM FeCl3) was mixed with an equal amount (0.6 l) of reservoir solution (100 mM Tris, pH 7.8, 1.8 M ammonium sulfate). A platelike crystal was flash-frozen in liquid nitrogen after having been swept through a reservoir solution with 10% (2R,3R)-(Ϫ)-2,3-butanediol, 5 mM myo-inositol, 1 mM L-cysteine, and 1 mM FeCl3. The data are essentially complete to 1.8 Å (statistics shown in square brackets in Table 1), but using higher resolution but very incomplete data in refinement enhanced model geometry and the quality of 2FO Ϫ FC maps; they were used.
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