4KJD

RatIntestinal AP expressed in E. coli

4KJD の概要

• エントリーDOI: 10.2210/pdb4kjd/pdb
• 関連するPDBエントリー: 4KJG
• 分子名称: Intestinal-type alkaline phosphatase 1, MAGNESIUM ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: alpha/beta fold, hydrolase
• 由来する生物種: Rattus norvegicus (brown rat,rat,rats)
• 細胞内の位置: Cell membrane; Lipid-anchor, GPI-anchor: P15693
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 106473.61

構造登録者

Ghosh, K.,Anumula, R.K.,Laksmaiah, B.K. (登録日: 2013-05-03, 公開日: 2013-10-16, 最終更新日: 2024-11-20)

主引用文献

Ghosh, K.,Mazumder Tagore, D.,Anumula, R.,Lakshmaiah, B.,Kumar, P.P.,Singaram, S.,Matan, T.,Kallipatti, S.,Selvam, S.,Krishnamurthy, P.,Ramarao, M.
Crystal structure of rat intestinal alkaline phosphatase - Role of crown domain in mammalian alkaline phosphatases.
J.Struct.Biol., 184:182-192, 2013

PubMed Abstract: 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 ~200-fold loss of activity that was partially recovered by the addition of external Zn(2+) and Mg(2+) 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 Zn(2+) and Mg(2+) whereas activity of most other alkaline phosphatases (APs) including the cIAP was strongly inhibited by excess Zn(2+). Based on our crystal structure, we hypothesized the residue Q317 in rIAP, 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 rIAP, where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity.

PubMed: 24076154
DOI: 10.1016/j.jsb.2013.09.017

実験手法

X-RAY DIFFRACTION (2.21 Å)