Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase

Abstract

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the Gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here we report the apo 2.1 Å resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.

Pyrrolysine Analogs as Substrates for Bacterial Pyrrolysyl-tRNA Synthetasein Vitroandin Vivo

“…5) As compared to archaeal PylRS, the amino acid residues associated with the substrate side-chain carbonyl group are conserved in DhPylRS, suggesting that DhPylRS also requires a carbonyl group at the side-chain amino group for substrate recognition. 4,11) Consistently with this, it has been demonstrated that DhPylRS also recognizes Lys(Cyc) as a substrate.…”

“…11,12) However, unlike M. barkeri PylRS, the substrate specificity of DhPylRS has not yet been well investigated, and only a few studies have been reported. 12) Recent protein engineering efforts make it possible to add non-canonical amino acids to the genetic code.…”

“…1. L-Lysine, Lys(Boc) (3), Lys(Ac) (4), Lys(Z) (5), Lys(Fmoc) (10), Lys(Aloc) (11), and Lys(Biot) (12) were purchased from commercial sources. The other Lys derivatives were chemically synthesized.…”

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

“…Expression of Genes and Purification of Products-The D. hafniense PylSc and M. barkeri PylS proteins were purified following recombinant expression of their genes as described previously (14,30). The D. hafniense pylSn gene was amplified by PCR from genomic DNA using Ex Taq (Takara Mirus Bio, Madison, WI) and primers PylSnF and PylSnR (the sequence of these and other primers are listed in supplemental Table S1).…”

“…The deletion was necessary to eliminate the highly basic yet hydrophobic N-terminal portion of the protein, whose presence interfered with protein stability and crystallization (29). The structure of D. hafniense PylSc is similar to that of M. mazei ⌬185PylS, although with an apparently tighter binding pocket for pyrrolysine (13,30). PylSc binds tRNA Pyl primarily through interactions with residues of the acceptor and D stem with no direct contact to residues of the T arm, anticodon arm, or variable loop (13).…”