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

Abstract

Mg-chelatase catalyzes the insertion of Mg 2þ into protoporphyrin IX at the first committed step of the chlorophyll biosynthetic pathway. It consists of three subunits: I, D, and H. The I subunit belongs to the AAA protein superfamily (ATPases associated with various cellular activities) that is known to form hexameric ring structures in an ATP-dependant fashion. Dominant mutations in the I subunit revealed that it functions in a cooperative manner. We demonstrated that the D subunit forms ATP-independent oligomeric structures and should also be classified as an AAA protein. Furthermore, we addressed the question of cooperativity of the D subunit with barley (Hordeum vulgare) mutant analyses. The recessive behavior in vivo was explained by the absence of mutant proteins in the barley cell. Analogous mutations in Rhodobacter capsulatus and the resulting D proteins were studied in vitro. Mixtures of wild-type and mutant R. capsulatus D subunits showed a lower activity compared with wild-type subunits alone. Thus, the mutant D subunits displayed dominant behavior in vitro, revealing cooperativity between the D subunits in the oligomeric state. We propose a model where the D oligomer forms a platform for the stepwise assembly of the I subunits. The cooperative behavior suggests that the D oligomer takes an active part in the conformational dynamics between the subunits of the enzyme.

Spark plasma sintering of commercial and development titanium alloy powders

“…17 MIDAS motifs frequently bind divalent metals and can be involved in protein− protein interactions, although these roles have not yet been demonstrated for the MIDAS motif in ChlD. 18 Six chimeric variants of ChlD were generated ( Figure 1).…”

Kinetic Analyses of the Magnesium Chelatase Provide Insights into the Mechanism, Structure, and Formation of the Complex

“…This subunit also appears to form hexamers observed with EM; however, this oligomerization does not require ATP (21). Biochemical data first demonstrated the likely formation of a BchI⅐BchD complex because the lag phase of the magnesium chelatase reaction was overcome by premixing of the BchI and BchD subunits in the presence of magnesium and ATP (17).…”

“…The enzyme from the latter three organisms was purified after heterologous expression of the subunits in Escherichia coli. Thus far, the most detailed magnesium chelatase and ATPase kinetic studies were performed with Synechocystis enzyme (17)(18)(19)(20), whereas the majority of recent structural information is from R. capsulatus using crystallization and electron microscopy (EM) analysis of individual subunits (21)(22)(23)(24)(25).…”

The Barley Magnesium Chelatase 150-kD Subunit Is Not an Abscisic Acid Receptor

“…In mutant xantha-f.58 the entire Xantha-f gene is deleted . In xantha-g.44, a GTT codon has been changed to CTT (V390 to L; Axelsson et al, 2006). Only a trace amount of XanG is found in xantha-g.44, which is a slightly leaky mutant with 7% of the chlorophyll found in the wild type (Henningsen et al, 1993).…”

“…In barley (Hordeum vulgare) the 40-, 70-, and 150-kD subunits are called XanH, XanG, and XanF, respectively, as they are encoded by the genes Xantha-h, -g, and -f (von Wettstein et al, 1971). The two smaller subunits form a double hexameric ring complex typical of an AAA protein (ATPase associated with various cellular activities; Fodje et al, 2001;Reid et al, 2003;Willows et al, 2004;Axelsson et al, 2006;Elmlund et al, 2008). The diverse AAA proteins are a superfamily involved in processes such as folding, assembly, and disassembly of protein complexes (Neuwald et al, 1999;Iyer et al, 2004).…”