General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing

Jing Zhao, Daniel A. Ciulla, Jian Xie, Andrew G. Wagner, Drew A. Castillo, Allison S. Zwarycz, Zhongqian Lin, Seth Beadle, José Luis Giner, Zhong Li, Hongmin Li, Nilesh Banavali, Brian P. Callahan, Chunyu Wang

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

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 in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 × 103 and 3.7 × 103 M-1 s-1, respectively, and an identical pKa = 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.

Original languageEnglish (US)
Pages (from-to)18380-18384
Number of pages5
JournalJournal of the American Chemical Society
Volume141
Issue number46
DOIs
StatePublished - Nov 20 2019
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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