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General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.
Zhao, Jing; Ciulla, Daniel A; Xie, Jian; Wagner, Andrew G; Castillo, Drew A; Zwarycz, Allison S; Lin, Zhongqian; Beadle, Seth; Giner, José-Luis; Li, Zhong; Li, Hongmin; Banavali, Nilesh; Callahan, Brian P; Wang, Chunyu.
Afiliación
  • Ciulla DA; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States.
  • Wagner AG; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States.
  • Castillo DA; Department of Chemistry , Binghamton University, State University of New York , Binghamton , New York 13902 , United States.
  • Beadle S; Department of Chemistry , State University of New York-ESF , Syracuse , New York 13210 , United States.
  • Giner JL; Department of Chemistry , State University of New York-ESF , Syracuse , New York 13210 , United States.
  • Li Z; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States.
  • Li H; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States.
  • Banavali N; Department of Biomedical Sciences, School of Public Health , University at Albany , Albany , New York 12222 , United States.
  • Callahan BP; Wadsworth Center , New York State Department of Health , Albany , New York 12201 , United States.
  • Wang C; Department of Biomedical Sciences, School of Public Health , University at Albany , Albany , New York 12222 , United States.
J Am Chem Soc ; 141(46): 18380-18384, 2019 11 20.
Article en En | MEDLINE | ID: mdl-31682419
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Colesterol / Proteínas Hedgehog Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: J Am Chem Soc Año: 2019 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Colesterol / Proteínas Hedgehog Tipo de estudio: Prognostic_studies Límite: Animals / Humans Idioma: En Revista: J Am Chem Soc Año: 2019 Tipo del documento: Article Pais de publicación: Estados Unidos