PDBsum entry 4ffr

Ligase

PDB id: 4ffr

Contents

Protein chain

347 a.a.

Ligands

ATP ×2

Metals

_MG ×4

Waters ×197

References listed in PDB file

Key reference

• Title: Biosynthesis of the 22nd genetically encoded amino acid pyrrolysine: structure and reaction mechanism of pylc at 1.5å resolution.
• Authors: F.Quitterer, A.List, P.Beck, A.Bacher, M.Groll.
• Ref.: J Mol Biol, 2012, 424, 270-282.
• PubMed id: 22985965

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

Abstract

The second step in the biosynthesis of the 22nd genetically encoded amino acid pyrrolysine (Pyl) is catalyzed by PylC that forms the pseudopeptide L-lysine-N(ε)-3R-methyl-D-ornithine. Here, we present six crystal structures of the monomeric active ligase in complex with substrates, reaction intermediates, and products including ATP, the non-hydrolyzable ATP analogue 5'-adenylyl-β-γ-imidodiphosphate, ADP, D-ornithine (D-Orn), L-lysine (Lys), phosphorylated D-Orn, L-lysine-N(ε)-D-ornithine, inorganic phosphate, carbonate, and Mg(2+). The overall structure of PylC reveals similarities to the superfamily of ATP-grasp enzymes; however, there exist unique structural and functional features for a topological control of successive substrate entry and product release. Furthermore, the presented high-resolution structures provide detailed insights into the reaction mechanism of isopeptide bond formation starting with phosphorylation of D-Orn by transfer of a phosphate moiety from activated ATP. The binding of Lys to the enzyme complex is then followed by an S(N)2 reaction resulting in L-lysine-N(ε)-D-ornithine and inorganic phosphate. Surprisingly, PylC harbors two adenine nucleotides bound at the active site, what has not been observed in any ATP-grasp protein analyzed to date. Whereas one ATP molecule is involved in catalysis, the second adenine nucleotide functions as a selective anchor for the C- and N-terminus of the Lys substrate and is responsible for protein stability as shown by mutagenesis.