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Abstract
Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5–6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an apo model of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5–6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5–6 and the imidodiphosphate and its associated Mg2+ are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.
Highlights
Three types of convergently evolved enzymes hydrolyze inorganic pyrophosphate (PPi): type I and type II soluble pyrophosphatases and membrane-integral pyrophosphatases
We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase
The top hits would be synthesized and tested in vitro, before restarting the in silico cycle of the compound design
Summary
Three types of convergently evolved enzymes hydrolyze inorganic pyrophosphate (PPi): type I and type II soluble pyrophosphatases (sPPases) and membrane-integral pyrophosphatases (mPPases). PPi is generated by at least 190 cellular reactions including DNA biosynthesis and aminoacyl-tRNA generation; tight control of PPi levels in the cell is crucial to prevent product inhibition of these reactions. sPPases are present in all cells and are primarily responsible for managing cellular PPi levels, with a kcat of $200–2000 sÀ1.6 In contrast, mPPases are slower (kcat of 3–20 sÀ1), only found in select species, and utilize the hydrolysis of PPi to pump ions across a membrane, generating an electrochemical gradient. Excluding multicellular animals, such as mammals, mPPases are found in organisms across all three domains of life. Plants and algae express mPPases in vacuolar membranes that, along with the vacuolar (V-type) Hþ-ATPase, acidify the organelle and are involved in development and stress resistance. mPPases are found in the membranes of the acidocalcisome, a relatively small organelle found in protozoan parasites that plays a crucial role during the transition between environments with varying osmotic pressures. mPPases are present in many bacterial species, several of which are opportunistic human pathogens, such as members of genus Bacteroides. Overexpression of mPPases in bacteria confers resistance to heat, hydrogen peroxide, and salt stress.
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