O -Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research.

Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNAPyl1. The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines2. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species1,3. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyzes the formation of Pyl-tRNAPyl4,5. Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor6; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code7,8. This technology relies on the directed evolution of an ‘orthogonal’ tRNA synthetase:tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNAPyl function as an orthogonal pair in vivo5,9. Here we demonstrate that Desulfitobacterium hafniense PylRS:tRNAPyl is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS:tRNAPyl allowed for the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS:tRNA interaction surface highly distinct from those observed in any other known aaRS:tRNA complex; it is this general property that underlies the molecular basis of orthogonality.

SUMMARY Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca2+ influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

a b s t r a c tPosttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published [1,2] AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.