Intestinal alkaline phosphatases (IAPs) are involved in the cleavage of phosphate prodrugs to liberate the drug for absorption in the intestine. To facilitate in vitro characterization of phosphate prodrugs, we have cloned, expressed, purified and characterized IAPs from rat and cynomolgus monkey (rIAP and cIAP respectively) which are important pre-clinical species for drug metabolism studies. The recombinant rat and monkey enzymes expressed in Sf9 insect cells (IAP-Ic) were found to be glycosylated and active. Expression of rat IAP in Escherichia coli (rIAP-Ec) led to similar to 200-fold loss of activity that was partially recovered by the addition of external Zr2+ and Mg2+ ions. Crystal structures of rIAP-Ec and rIAP-Ic were determined and they provide rationale for the discrepancy in enzyme activities. Rat IAP-Ic retains its activity in presence of both Zn2+ and Mg2+ whereas activity of most other alkaline phosphatases (APs) including the cIAP was strongly inhibited by excess Zn2+. Based on our crystal structure, we hypothesized the residue Q317 in rIAP, present within 7 angstrom of the Mg2+ at M3, to be important for this difference in activity. The Q317H rIAP and H317QcIAP mutants showed reversal in effect of Zn2+, corroborating the hypothesis. Further analysis of the two structures indicated a close linkage between glycosylation and crown domain stability. A triple mutant of rIAP, where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity. (C) 2013 Elsevier Inc. All rights reserved.