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

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 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 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 orthologs 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.