Figure 4: Mutagenesis studies to assess the structural observations.
From: A distinct holoenzyme organization for two-subunit pyruvate carboxylase
(a) Interactions between the BT-like domain and the BC dimer at the holoenzyme interface. (b) Overlay of the structure of MfPC (in colour) and SaPC (in grey) in complex with CoA (grey for carbon atoms)10. The red arrow indicates the difference in position of the BT-like domain in MfPC and the PT domain in SaPC. (c) Catalytic activities of wild-type and mutant MfPCs. The pyruvate concentration is at 20 mM. The error bars represent the s.d. from three independent measurements. NA, no activity observed under the condition tested. (d) Deletion of the α helix of the BT-like domain, or mutation of one of its Ala residues (A465R), abolished the formation of the MfPC holoenzyme. The two subunits were co-expressed in E. coli, with the α subunit carrying a His-tag. E, eluate from the Nickel column; F, flow-through of the Nickel column; P, pellet; S, soluble fraction. (e) Mutation of an Ala residue (A465R) in the helix of the BT-like domain abolished the formation of the MfPC holoenzyme. (f) A quadruple mutation in the interface between the BT-like domain and BC blocked holoenzyme formation.
