CDNA Cloning and Characterization of Enzymes That Synthesize Bile Acids, Vitamin D and Waxes

Countless enzymes are required for the synthesis of the diverse array of lipids found in nature. The identification and characterization of five different lipid metabolizing enzymes are reported here. The 3beta -hydroxy-delta 5-C27-steroid oxidoreductase (C27 3beta-HSD) enzyme catalyzes a step in bile acid synthesis. Subjects with mutations in the encoding gene fail to synthesize bile acids and develop liver disease. Fifteen patients were screened and twelve different mutations were identified in the C27 3beta -HSD gene. Vitamin D is required for normal bone metabolism and maintenance of serum calcium levels. The conversion of vitamin D into an active ligand requires 25-hydroxylation. I report here the identification by expression cloning of a cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. A patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency was identified. Molecular analysis of this individual revealed homozygosity for a transition mutation in the CYP2R1 gene causing the substitution of a proline for a leucine in the protein and eliminating vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. The reduction of fatty acids to fatty alcohols, by a fatty acyl-CoA reductase enzyme, is required for the synthesis of wax monoesters and ether lipids. Using a bioinformatics approach, the first two mammalian fatty acyl-CoA reductase genes (FAR1 and FAR2) were identified. The two mouse FAR enzymes, which share 57% sequence identity at the amino acid level, have differing substrate specificities and tissue distributions implying unique physiological roles for each. Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. Here, I report the identification by expression cloning of a wax synthase gene. Co-expression of cDNAs specifying FAR1 and wax synthase led to the synthesis of wax monoesters. The data suggests that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes.