Posttranslational modifications are essential for the biological function of many proteins. Prenylation is one such modification in which protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify their substrates by attachment of a 20- or 15- carbon isoprenoid moiety to a cysteine residue near the C-terminus of their substrates. These enzymes recognize their substrates based on the presence of a 'Ca1a2X' motif near the C- terminus where C denotes the cysteine residue that is prenylated and a1, a2 and X act as substrate recognition elements for FTase and/or GGTase-I. While the a2 residue binding pocket within GGTase-I has been characterized structurally, the functional impact of enzyme-substrate interactions in the active site remains to be determined. Using site-directed mutagenesis, targeted evolution and peptide substrate structure-activity studies, we identified amino acid residues in the active site of GGTase-I that play an important role in substrate specificity. GGTase-I substrate specificity can be tuned by mutating specific amino acids in the active site, much like FTase. Our work suggests that tunable substrate specificity may be a general property of multispecific enzymes involved in posttranslational modification.  The GGTase-I variants developed in this work can be used to create bioengineered prenylation pathways inside the cell, which can potentially probe the effects of prenylation pathway modifications on protein localization and function without global perturbation of the prenylated proteome.

Development of novel GGTase-I variants with altered substrate specificity through targeted active site reengineering

248th National Meeting (2014)

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