Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase

Abstract

Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R-->A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R-->A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F- inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+-PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

“…Mung bean (V. radiata L.) H ϩ -PPase cDNA (VPP; accession number P21616) was cloned into the Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2 (Invitrogen), and the C terminus was fused with His 6 tag (pYVH6) for purification and reconstitution (7). The site-directed mutagenesis was carried out by the QuikChange TM method with the primers containing mutated oligonucleotides (Table 1) and then subcloned to the expression vector pYVH6 (13,25). The mutated nucleotides were subsequently defined and confirmed by DNA sequencing.…”

“…The pYVH6 and its relative variants were transformed into the yeast host cell S. cerevisiae strain BJ2168 (MATa, prc-407, prb1-1122, pep4-3, leu2, trp1, ura3, GAL) and cultured according to previous methods (7,12,13). The yeast microsomal membranes enriched in H ϩ -PPases were prepared followed by solubilization and purification with Ni 2ϩ -nitrilotriacetic acid beads as described previously (7,12,13,25).…”

Identification of Essential Lysines Involved in Substrate Binding of Vacuolar H+-Pyrophosphatase

“…ϩ -PPase in Yeast Cells-Vacuolar H ϩ -PPase cDNA of Vigna radiata (GenBank TM accession number AB009077) was cloned and inserted into pBlueScript II SK(ϩ) between the HindIII and XbaI cutting sites for DNA preparation and manipulation as described previously (14). The site-directed mutagenesis was carried out by the PCR megaprimer method (26) with the primer containing mutated oligonucleotides (supplemental Table S1).…”

“…Furthermore, Mg 2ϩ is essential for the activity of H ϩ -PPase, whereas Ca 2ϩ is inhibitory as it prevents the formation of the substrate complex. Fluoride is also an H ϩ -PPase inhibitor (12)(13)(14)(15)(16)(17). H ϩ -PPases from different species have ϳ30% sequence identity overall, and their conserved residues are clustered in three cytosolic loops, i.e.…”

“…A sulfhydryl-modifying reagent, mersalyl, attacked the substrate-protectable cysteines of Rhodospirillum rubrum H ϩ -PPase (22). His-716 of mung bean H ϩ -PPase was the target of the noncompetitive inhibitor, diethyl pyrocarbonate (23,24), and Arg-242 has been recently identified as a substrate-binding residue (14).…”