Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition

Biochemistry. 2014 Jan 21;53(2):434-46. doi: 10.1021/bi4011732. Epub 2014 Jan 6.


Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca1a2X sequence on their substrates, with the a1, a2, and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a2 residue binding pocket, consistent with a preference for moderately sized a2 residues in GGTase-I substrates, the functional impact of enzyme-substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure-activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues. Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / chemistry
  • Alkyl and Aryl Transferases / genetics
  • Alkyl and Aryl Transferases / metabolism*
  • Animals
  • Biocatalysis
  • Catalytic Domain
  • Chromatography, High Pressure Liquid
  • Humans
  • Models, Molecular
  • Molecular Structure
  • Peptide Library
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Engineering*
  • Rats
  • Substrate Specificity


  • Peptide Library
  • Peptides
  • Alkyl and Aryl Transferases
  • geranylgeranyltransferase type-I