Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA ⁺ hexamers

Abstract

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg 2؉ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA ؉ ) family of proteins. AAA ؉ proteins are Mg 2؉ -dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.ATPase ͉ chlorophyll biosynthesis ͉ integrin ͉ metallation ͉ sensor arginine

Recessiveness and Dominance in Barley Mutants Deficient in Mg-Chelatase Subunit D, an AAA Protein Involved in Chlorophyll Biosynthesis

“…This inhibitory or poisoning effect of the mutant I subunits on the wild-type I subunits was >50% at a 1:1 ratio in the R. capsulatus system, whereas a threefold to ninefold excess of mutant subunits over wild-type protein was required to obtain a 50% inhibition in Synechocystis. These experiments demonstrated the dominant effect of the proteins in vitro, and it was concluded that the I subunits function in a concerted manner within the hexameric complex (Hansson et al, 2002).…”

“…Previous analyses of semidominant and recessive barley and maize (Zea mays) Mg-chelatase mutants deficient in the I subunit have also provided knowledge on the interactions between the subunits (Hansson et al, , 2002Sawers et al, 2006). The D subunit could not be detected in any of the studied barley I mutants, whereas wild-type levels of the D subunit were found in barley mutants that lacked the H subunit (Lake et al, 2004).…”

“…Thus, the recessive xantha-h mutants contained no I protein, whereas wild-type levels were found in the semidominant mutants. For in vitro studies, the semidominant mutations found in barley and maize were constructed in existing expression systems for R. capsulatus bchI and Synechocystis chlI, respectively (Hansson et al, 2002;Sawers et al, 2006). The resulting I proteins showed no or reduced ATPase activity and could not contribute to the Mg-chelatase activity.…”

Suppression of the Barleyuroporphyrinogen III synthaseGene by aDsActivation Tagging Element Generates Developmental Photosensitivity

“…Unlike the four preceding enzymatic steps ( Figure 1A), reduced Mg-chelatase activity does not cause photosensitivity, but rather reduced chlorophyll levels due to a negative feedback regulatory mechanism (Hansson et al, 1999(Hansson et al, , 2002Papenbrock et al, 2000aPapenbrock et al, , 2000bSoldatova et al, 2005;Sawers et al, 2006). The substrate of Mg-chelatase, protoporphyrin IX, is the branch point of chlorophyll and heme synthesis ( Figure 1A); by contrast, antisense suppression of the first committed heme synthetic enzyme, plastidic ferrochelatase, does lead to protoporphyrin IX accumulation and light-dependent necrosis (Papenbrock et al, 2001).…”

Effect of Qingyitang on activity of intracellular Ca2+-Mg2+-ATPase in rats with acute pancreatitis

“…This finding suggested that intracellular Ca 2+ -Mg 2+ -ATPase activity was not only dependent on the level of its gene expression but also was affected by many other factors [17][18][19] .…”