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  • br Materials and methods br Acknowledgments br Introduction

    2019-09-11


    Materials and methods
    Acknowledgments
    Introduction Proton pumps convert the energy of high-energy compounds into the 1295 and of protons across biomembranes. They play two physiological roles: pH regulation and the formation of proton-motive forces in their membranes. The simplest of these pumps is the H+-translocating inorganic pyrophosphatase (H+-PPase), which consists of a single polypeptide of ∼80 kDa [1]. H+-PPases are found in plants, parasitic and free-living protozoa, and some eubacteria and archaebacteria [2], [3], [4], [5], [6], [7]. In prokaryotes, such as Rhodospirillum rubrum[2], [8], [9], Pyrobaculum aerophilum[10], and Agrobacterium tumefaciens[11], the enzyme generates a proton gradient across the plasma membrane and the membranes of acidocalcisomes. Its physiological role has also been investigated in plants [12], [13], [14], [15], [16] and other organisms [8], [17], [18], [19]. The H+-PPase enzyme is an excellent model for research into the coupling between pyrophosphate (PPi) hydrolysis and active proton transport because it consists of a single protein and has a simple substrate. Site-directed mutagenesis revealed several functional motifs in the H+-PPases of Arabidopsis thaliana[4], [20], [21], Vigna radiata[22], [23], [24], R. rubrum[25], [26], Carboxydothermus hydrogenoformans[27], and Streptomyces coelicolor A3(2) [28], [29], [30], [31]. Membrane topological analysis showed that all the common functional motifs for Mg-PPi binding and PPi hydrolysis are faced to the cell cytosol, which is where the substrate is generated [28]. In the present study, we investigated how H+-PPase couples the hydrolysis of PPi with the active transport of protons across the membrane. As a model enzyme we used S. coelicolor A3(2) H+-PPase (ScPP), which comprises 794 amino acids and 17 transmembrane domains (TMs) [28]. To examine the coupling mechanism and the structural–functional relationship, we separated the primary structure into four parts and constructed ScPP mutant libraries for each part using random mutagenesis. We focused on the third quarter, which consists of TM10 to TM13 and two conserved motifs in a hydrophilic loop. We prepared a random mutant library of H+-PPase, and surveyed mutants with a low coupling efficiency between PPi hydrolysis and proton pumping. We also prepared site-directed mutants of H+-PPase and determined the effect of mutating residues in the TMs and hydrophilic loops on enzyme activity and energy coupling. The structural and functional significance of these mutations is discussed in relation to the energy-transducing mechanism.
    Materials and methods
    Results
    Discussion To elucidate the proton transport pathway of H+-PPase, and the coupling mechanism between PPi hydrolysis and H+ translocation, we performed a series of random mutagenesis analysis of the H+-PPase of S. coelicolor A3(2) H+-PPase. Site-directed mutagenesis has successfully determined the essential residues for the binding and hydrolysis of the substrate, and has provided critical information on the structure–function relationship of the cytoplasmic catalytic domain and a few TMs [3], [4], [20], [22], [23], [24], [25], [26], [27], [28], [38], [39], [40]. Site-directed mutagenesis is effective for evaluating specific residues and random mutagenesis is also effective for finding unexpected functional residues. In the present study, we focused the third quarter region (Leu384 to Arg586) of ScPP and analyzed by random and site-directed mutagenesis. The third quarter region has highly conserved motifs in cytoplasmic loop k, which have been demonstrated to be essential for PPase activity by site-directed mutagenesis [3], [4]. Random mutagenesis revealed several unexpected residues that were involved in active proton transport and energy transfer. The results of the random and site-directed mutagenesis are summarized in the helical wheels (Fig. 6) and the membrane topology model (Fig. 7). These functional residues were found in the cytoplasmic loops, periplasmic (intravacuolar) loops, and TMs. We discuss them separately below.