Extended Data Fig. 2: Rational mutagenesis of PbCfaL.
From: Discovery, characterization and engineering of ligases for amide synthesis
a, Structural comparison between PbCfaL (left) and the mutant PbCfaL(R395G) (right). R395 (circled) of PbCfaL (PDB ID 7A9I) is in the hinge region between the N-terminal domain (blue) and the flexible C-terminal domain (red). In PbCfaL(R395G) (PDB ID 7A9J) this large arginine residue is replaced by a much smaller glycine (circled) that is found in the other members of the CfaL family and many other similar ANL ligases. The overall structure of this mutant exhibits no other substantial structural difference from that of the wild type. b, Overlay of PbCfaL with three published ATP-dependent ligase structures (in ellipse) showing the conserved ATP binding location. When superimposed, PbCfaL (PDB ID 7A9I, blue), McbA (AMP bound, PDB ID 6SQ8, red), GrsA (ATP bound, PDB ID 1AMU, green) and AuaEII (anthranoyl-AMP bound, PDB ID 4WV3, light brown) show the conserved location of ATP binding. The corresponding loop in PbCfaL (inset, arrowed) is larger than in the other structures which may affect ATP binding. The location of this region within the structure of PbCfaL (grey) is also shown for reference. Structural alignment was performed using Chimera (version 1.14) MatchMaker.
