Nine of Ten Histidine Residues, Located in Three Conserved Clusters, are Required for Mammalian Sterol C5-Desaturase Activity

Abstract

We previously reported three histidine-rich conserved motifs containing HX3H, HX2H and HX2HH in the deduced primary amino acid sequence of human sterol C-5 desaturase (SC5D), which introduces a double bond at the C5, 6 positions of A7-sterols to form A5,7-sterols (Nishi, S. et al. (2000) Biochim. Biophys. Acta 1490, 106-108). In the present study, the role of the 18 His residues contained in the enzyme was investigated by means of site-directed mutagenesis and expression of the mutated enzymes in an SC5D-deficient yeast strain (erg3). The activity of the recombinant SC5Ds to allow the yeast to convert ergosta-7,22-dien-3β-ol to ergosterol (ergosta-5,7,22-trien-3β-ol) was examined directly in the erg3 strain in vivo. Individual replacements of each of the ten conserved His residues with Arg completely eliminated the desaturase activity in each case, except for one, His190. The conserved His-containing clusters have a consistent positioning with respect to the putative membrane spanning domains, and we propose that they are located on the cytoplasmic face of the endoplasmic reticulum. In contrast, mutation of eight nonconserved flanking His residues to Arg did not affect the ability to complement the erg3 phenotype. Therefore, these non-conserved His residues are not essential for the catalysis, but may contribute to the activity through conformational or other effects. Western blot analysis confirmed that the steady-state expression levels were equivalent for the wild-type human SC5D and all the mutants, supporting the idea that the conserved His residues are essential for catalytic function. A possible role for these His residues could be to act as ligands for the iron atom(s) contained in the enzyme. Hydropathy analyses indicated that the enzyme contains at least four membrane-spanning domains. Topologically, SC5D is predicted to have a C-terminal cytoplasmic domain and an N-terminal region located in the luminal side of the endoplasmic reticulum, since the N-terminal sequence was resistant to proteinase K proteolysis, but the C-terminal sequence was not.