Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus

Abstract

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.

CHLH/GUN5 Function in Tetrapyrrole Metabolism Is Correlated with Plastid Signaling but not ABA Responses in Guard Cells

“…It was reported that CHLH predominantly exists as a monomer in solution (Qian et al, 2012), whereas a loosely bound CHLH dimer was observed in the crystal. The dimerization interface are domains I and V, which is consistent with a previous study that removal of the N-terminal 159 residues of T. elongates ChlH facilitates a monomeric state (Adams et al, 2014). The porphyrin-binding internal pocket is proposed to be located at the interface between domains III and V, a region with the most conserved residues.…”

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

“…It also appears that Gun4 proteins from virtually all sources stimulate MgCh activity via an interaction with the ChlH subunit and bind porphyrins with the highest affinity to MgP (14,16,17). The essential role of porphyrin binding for Gun4 function can be suggested from amino acid alignments (13) because residues in the proposed MgP binding site (␣6/␣7 loop residues, Arg-214, and Asn-211) are conserved through evolution from cyanobacteria to plants.…”

“…There is evidence that Gun4 physically interacts with ChlH (11,14,17), which is the porphyrin-binding MgCh subunit that presumably also contains the active site for chelation (18). Available in vitro kinetic data imply that Gun4 stimulates MgCh by substantially reducing the P IX and Mg 2ϩ concentrations required for catalysis (12).…”

“…Functional MgCh enzyme assays were performed essentially as described previously (17). Calibration curves using known standard concentrations of magnesium deuteroporphyrin IX (MgD) were created for each concentration of Gun4 analyzed.…”

The active site of magnesium chelatase

“…MgCHs require at least three subunits; chlorophyll producing organisms have ChlI (~35 kDa), ChlD (~75 kDa) and ChlH (~150 kDa), and the closely related proteins from bacteriochlorophyll producing organisms are BchI, BchD, and BchH 7, 8 . In cyanobacteria and higher plants a fourth regulatory subunit, Gun4, is required for full protein activity [9][10][11] .The genes for MgCH were originally identified and recombinant protein expression systems were developed some time ago 7,12 , and extensive kinetic characterization of the MgCH has identified the roles of the subunits [13][14][15][16][17][18] . The current mechanistic and structural data suggest a model for the MgCH mechanism where the two AAA + subunits form the ChlID complex 16,19 .…”