Structural and Biochemical Characterization of Gun4 Suggests a Mechanism for Its Role in Chlorophyll Biosynthesis^,

Abstract

Gun4 has been implicated in a developmental signaling pathway between the chloroplast and the nucleus involving magnesium protoporphyrin IX (MgP(IX)), the first dedicated intermediate in the chlorophyll biosynthetic pathway. Here we present the crystal structure of Thermosynechococcus elongatus Gun4 at 1.5 A, describe the binding affinities of Gun4 for substrate and product porphyrin molecules, and identify a likely (Mg)P(IX) binding site on the protein. Kinetic analyses show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product and that it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. Gun4 therefore controls magnesium chelatase at physiologically significant Mg2+ concentrations and likely acts as a molecular switch in vivo so that in its absence magnesium chelatase is inactive. This mechanism could allow Gun4 to mediate magnesium protoporphyrin levels both for chlorophyll biosynthesis and for signaling to the nucleus.

Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis

“…Plant and cyanobacterial mutants in gun4 display reduced Chl content and impaired growth, and they accumulate the substrate for MgCH, whereas phototrophic bacteria lack orthologs of gun4 (34). Gun4 was found to stimulate cyanobacterial MgCH activity in vitro (35) and to be involved in increasing flux into the Chl biosynthesis pathway in vivo (36). Similarly, it is conceivable that Ycf54 may play a role in substrate channeling; pull-down experiments identified protein-protein interactions between Ycf54 and the cyanobacterial AcsF, CycI (21), and CycI with other Chl biosynthesis enzymes (22).…”

“…When required, kanamycin was added at 50 μg·mL −1 . Synechocystis strains were grown photoautotrophically in a rotary shaker at 30°C under low light (5 μmol photons m −2 ·s −1 ) or moderate light (30 μmol photons m −2 ·s −1 ) as described previously (35). Photomixotrophic growth was achieved by the addition of glucose to a final concentration of 5 mM.…”

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

“…Tetrapyrrole biosynthesis ranks among the most fundamental pathways in living systems, leading to production of heme, siroheme, and vitamin B 12 . Synthesis of heme is conserved among all three domains of life, starting with synthesis of ␦-aminolevulinic acid (ALA), 4 which is converted through several enzymatic steps into protoporphyrin IX (P IX ), a metal-free porphyrin ring.…”

“…In bacteriochlorophyll-producing bacteria, these three subunits are sufficient for a highly active MgCh enzyme when assayed in vitro (10). In contrast, MgCh from cyanobacteria, algae, and plants requires a protein called Gun4 that strongly enhances MgCh activity in vitro (11)(12)(13) and very likely also in vivo (14,15).…”

“…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). Inactivation of the gun4 gene perturbs Chl biosynthesis and accumulation of Chl-binding proteins in cyanobacteria (14), green algae (15), and plants (11,19).…”