6TYY

Hedgehog autoprocessing mutant D46H

6TYY の概要

• エントリーDOI: 10.2210/pdb6tyy/pdb
• 分子名称: Protein hedgehog (2 entities in total)
• 機能のキーワード: hedgehog autoprocessing doamin, signaling protein
• 由来する生物種: Drosophila melanogaster (Fruit fly)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 15999.28

構造登録者

Li, H.,Li, Z.,Wang, C.,Callahan, B.P. (登録日: 2019-08-09, 公開日: 2019-11-20, 最終更新日: 2024-11-20)

主引用文献

Zhao, J.,Ciulla, D.A.,Xie, J.,Wagner, A.G.,Castillo, D.A.,Zwarycz, A.S.,Lin, Z.,Beadle, S.,Giner, J.L.,Li, Z.,Li, H.,Banavali, N.,Callahan, B.P.,Wang, C.
General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.
J.Am.Chem.Soc., 141:18380-18384, 2019

PubMed Abstract: Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H using a continuous FRET-based autoprocessing assay and with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with / = 2.1 × 10 and 3.7 × 10 M s, respectively, and an identical p = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

PubMed: 31682419
DOI: 10.1021/jacs.9b08914

実験手法

X-RAY DIFFRACTION (1.36 Å)