title: Expansion of protein farnesyltransferase specificity using "tunable" active site interactions: development of bioengineered prenylation pathways.
abstract: Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be "tuned" using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA. hougland@syr.edu
authors.forename: James L
authors.initials: JL
authors.lastname: Hougland
authors.forename: Soumyashree A
authors.initials: SA
authors.lastname: Gangopadhyay
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D021122:Protein Subunits; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase