Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

Background: FTase recognizes and modifies many proteins with C-terminal CA1A2X sequences. Results: Mutating active site residues Trp-102β and Trp-106β significantly alters FTase peptide selectivity both in vitro and in vivo. Conclusion: FTase substrate selectivity includes negative discrimination that can be relaxed/altered without losing activity. Significance: Deciphering FTase peptide recognition allows creation of bioengineered prenylation pathways and provides a model for other multispecific enzymes. 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.

• Publication year

  2012

• Venue

  Journal of Biological Chemistry

• Publication date

  2012-09-19

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.M112.404954PMID 22992747PMCID PMC3488079
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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