6TYY

Hedgehog autoprocessing mutant D46H

Summary for 6TYY

• Entry DOI: 10.2210/pdb6tyy/pdb
• Descriptor: Protein hedgehog (2 entities in total)
• Functional Keywords: hedgehog autoprocessing doamin, signaling protein
• Biological source: Drosophila melanogaster (Fruit fly)
• Total number of polymer chains: 1
• Total formula weight: 15999.28

Authors

Li, H.,Li, Z.,Wang, C.,Callahan, B.P. (deposition date: 2019-08-09, release date: 2019-11-20, Last modification date: 2024-11-20)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.36 Å)