Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Ring-forming AAA؉ ATPases act in a plethora of cellular processes by remodeling macromolecules.

SummaryIn plants, the first committed enzyme for glutathione biosynthesis, c-glutamylcysteine ligase (GCL), is under multiple controls. The recent elucidation of GCL structure from Brassica juncea (BjGCL) has revealed the presence of two intramolecular disulfide bridges (CC1, CC2), which both strongly impact on GCL activity in vitro. Here we demonstrate that cysteines of CC1 are confined to plant species from the Rosids clade, and are absent in other plant families. Conversely, cysteines of CC2 involved in the monomer-dimer transition in BjGCL are not only conserved in the plant kingdom, but are also conserved in the evolutionarily related a-(and some c-) proteobacterial GCLs. Focusing on the role of CC2 for GCL redox regulation, we have extended our analysis to all available plant (31; including moss and algal) and related proteobacterial GCL (46) protein sequences. Amino acids contributing to the homodimer interface in BjGCL are highly conserved among plant GCLs, but are not conserved in related proteobacterial GCLs. To probe the significance of this distinction, recombinant GCLs from Nicotiana tabacum (NtGCL), Agrobacterium tumefaciens (AtuGCL, a-proteobacteria) and Xanthomonas campestris (XcaGCL, c-proteobacteria) were analyzed for their redox response. As expected, NtGCL forms a homodimer under oxidizing conditions, and is activated more than threefold. Conversely, proteobacterial GCLs remain monomeric under oxidizing and reducing conditions, and their activities are not inhibited by DTT, despite the presence of CC2. We conclude that although plant GCLs are evolutionarily related to proteobacterial GCLs, redox regulation of their GCLs via CC2-dependent dimerization has been acquired later in evolution, possibly as a consequence of compartmentation in the redox-modulated plastid environment.

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