title: Targeted reengineering of protein geranylgeranyltransferase type I selectivity functionally implicates active-site residues in protein-substrate recognition.
abstract: 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.
authors.affiliation: Department of Chemistry, Syracuse University , Syracuse, New York 13244, United States.
authors.forename: Soumyashree A
authors.initials: SA
authors.lastname: Gangopadhyay
authors.forename: Erica L
authors.initials: EL
authors.lastname: Losito
authors.forename: James L
authors.initials: JL
authors.lastname: Hougland
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D013379:Substrate Specificity
chemical_list: D019151:Peptide Library; D010455:Peptides; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I