Title : Crystal structure of rat
intestinal alkaline phosphatase--role of crown
domain in mammalian alkaline phosphatases
Abstract :
- Intestinal alkaline phosphatases (I APs ) 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 I APs from rat and cynomolgus monkey (r IAP and c IAP 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 (r IAP-Ec) led to ~200-fold loss of activity that was partially recovered by the addition of external Zn(2+) and Mg(2 +) ions
- Crystal structures of r IAP-Ec and r IAP-Ic were determined and they provide rationale for the discrepancy in enzyme activities
- Rat IAP-Ic retains its activity in presence of both Zn(2+) and Mg(2 +) whereas activity of most other alkaline phosphatases ( APs ) including the c IAP was strongly inhibited by excess Zn(2+)
- Based on our crystal structure, we hypothesized the residue Q317 in r IAP , present within 7 Å of the Mg(2 +) at M3, to be important for this difference in activity
- The Q317H rIAP and H317Q cIAP mutants showed reversal in effect of Zn(2+), corroborating the hypothesis
- Further analysis of the two structures indicated a close linkage between glycosylation and crown domain stability
- A triple mutant of r IAP , where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity
Output (sent_index, trigger,
protein,
sugar,
site):
- 3. glycosylated, , enzymes, -, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):