Structure of the Mg-Chelatase Cofactor GUN4 Reveals a Novel Hand-Shaped Fold for Porphyrin Binding
Figure 7
Quantitative Analysis of the GUN4 Stimulation of Mg2+ Incorporation into Deutero
(A) The Km values for GUN4 assisted Mg-Deutero biosynthesis were determined using both 0.2 μm (red) and 0.4 μM SynGUN4 (green). The resultant values contrast with those obtained for Mg2+ incorporation by the Mg-chelatase complex in the absence of SynGUN4 (blue).
(B) Relative Km values (compared to wild-type SynGUN4) were determined for each SynGun4 mutant previously examined for Deutero binding.
(C) Rendered ribbon diagrams of orthogonal views of the SynGUN4 core domain with the relative Km values of each SynGUN4 mutant mapped onto the structure. The color-coded scale for each mutation's effect on Mg2+ incorporation is shown at the bottom. Several mutants that altered Mg2+ incorporation activity but previously did not affect binding to deuteroporphyrin IX were uncovered including a R217A mutation and the Trp129 and Tyr131 residing on the α2/α3 loop. Mutants of Val218, the later of which is critical for binding Deutero but not for binding Mg-Deutero showed no effect on chelatase activity while mutants of Ala219, the later of which is essential for binding to Mg-Deutero, completely failed to stimulate Mg-chelatase activity. Only those mutant SynGUN4s exhibiting a greater than 10-fold change in Km are labeled. Shown in black are residues that, when mutated to alanine, failed to produce properly folded protein upon expression in E. coli.