Aspartyl 3-hydroxylase (A3H; EC 1.14.11.16) generates hydroxyaspartate/hydroxyasparagine residues in a specific sequence of the epidermal growth factor (EGF) modules of extracellular proteins, among them Factor IX, complement subcomponent Cls, the fibrillins, and the latent transforming growth factor-binding proteins (LTBPs). The modified residues form very high affinity Ca2+ binding sites. In proteins containing clusters of EGF modules, like the fibrillins or the LTBPs, these binding sites determine the folding of unique helical structures and thus, their biological function; for instance, mutational loss of a single hydroxylated moiety among the 2871 residues of fibrillin-1 causes Marfan syndrome (Handfort et al., JBC 270, 6751-6756;1995). We have established an in vitro assay for human A3H, which we recombinantly expressed in its native and a truncated form as fusion protein, and analyzed the domain structure of A3H. The cDNA encoding human A3H was isolated from an osteosarcoma cDNA library as described (Korioth et al., Gene 150, 395-399; 1994). For directed cloning into pGEM-3z, a novel BamHI site was engineered by PCR i) at the 5′- end, to generate the full-length product of 2274 nt, which encodes the native enzyme (NE) of residues 1-757; and ii) at nt 1045, to generate a truncated form (TF) of 1229 nt, which encodes a C-terminal fragment of residues 349-757. The NE and TF cDNAs each were ligated in frame into the appropriate pBlueBacHis™ transfer vectors, used to recombinantly generate baculovirus containing either the human NE or the human TF. Each protein, designed to contain a short N-terminal polyhistidine appendage for purification by chelation chromatography, was then expressed in baculovirus-infected High Five™ insect cells under serum-free conditions. To obtain underhydroxylated substrate, mouse C2C12 myoblasts expressing human Factor IX (Hortelano et al., Blood 87, 5095-5103; 1996) were incubated with the A3H inhibitor deferiprone. NE/TF activity was assessed by hydroxyaspartate formation using gas chromatography-mass spectrometry, and by cosubstrate consumption, i.e., 2-oxoglutarate decarboxylation. Surprisingly, TF was enzymatically active, despite truncation to just 53% of NE. The C-terminally located active site of human A3H apparently forms a functionally independent domain. Its computational analysis revealed 8 previously unrecognized sequence motifs shared with the C-terminus of human prolyl 4-hydroxylase (EC 1.14.11. 2). We propose that Cys 684, contained in one of these motifs, constitutes as thiolate the critical axial ligand of the ferrous metal center, in analogy to the oxygen-utilizing cytochrome P-450s. Our findings open an inroad into the molecular pathology of human A3H and its anticipated role in pediatric diseases, e.g. in cases of Marfan syndrome without apparent mutations in the fibrillin genes.