Evolutionary analysis of energy-converting enzymes

ATP synthase is the only known enzyme that can use the electrochemical potential of Na⁺ or H⁺ ions on the membrane to synthesize ATP molecules. Moreover, not all subunits of this enzyme are homologous in its different versions, and therefore evolutionary history of the enzyme is debatable. Investigations on the evolution of this enzyme (Mulkidjanian et al., 2007) and comparative structural and phylogenetic analysis showed that the sodium-binding site consisting of five residues in the membrane c/K -subunits of bacterial and archaeal enzymes is homologous and existed in the ancestral form of this enzyme, which means that the ancestral enzyme was sodium-translocating (Mulkidjanian et al., 2008). We have described a new subfamily of N-ATPases distributed by horizontal transfer and occupying a basal position on the phylogenetic tree of F-type ATP synthases, i.e. being evolutionarily early (Dibrova et al., 2010).

Modern cell membranes have a complex structure, and the biochemical synthesis of their lipid components includes many stages, many of which are fundamentally different in bacteria and archaea — respectively, lipids themselves are also different. The membranes of the Last Universal Cellular Ancestor (LUCA), and especially the primitive membranes of the first cells, were likely not strong enough to maintain the proton transmembrane potential (Mulkidjanian et al., 2009). As a result of the phylogenomic analysis of the enzymes responsible for the biosynthesis of fatty acids and their β-oxidation, we came to the conclusion that the widespread understanding of the antiquity of these processes is wrong (Dibrova et al., 2014). The issue of LUCA membranes remains open.

Based on the fundamental property of living cells to maintain strongly non-equilibrium concentration of K⁺ and Na⁺ ions inside the cell (i.e. to have the ratio K⁺ >> Na⁺ while in the external aquatic environment even in fresh water the ratio is the opposite), we assumed that life could appear in Charles Darwin’s imaginary “warm little pond,” on terrestian anoxic geothermal fields (Mulkidjanian et al., 2012). In addition to the predominance of the concentration of K⁺ over Na⁺, our “cradle of life” contains suitable concentrations of other ions, in particular zinc, which corresponds to several early hypotheses on the importance of zinc for early evolution living (Mulkidjanian et al., 2009; Mulkidjanian and Galperin, 2009; Mulkidjanian and Galperin, 2010). Location on the earth's surface, under the sunlight, also corresponds to the concept of resistance to ultraviolet radiation as an important evolutionary factor in the very early stages of prebiological evolution (Mulkidjanian et al., 2003). We suggest that, from an evolutionary point of view, membrane energy enzymes could arise from systems supporting sodium-potassium homeostasis, which in turn arose as a result of leaving the “cradle of life” for fresh and then salt water due to the need to maintain just such ratios of potassium and sodium ions which are vital for basic universal biochemical processes in the cell, especially for the protein synthesis (Dibrova et al., 2015).

The cytochrome bc complex is a widespread enzyme that generates a proton transmembrane potential due to the oxidation of quinol to quinone. Unlike many other energy-converting complexes, this enzyme is strictly proton due to the mechanism of its work on the principle of the “Mitchell loop”. Earlier, some scientists suggested that this enzyme is evolutionarily ancient and can be dated back to the time of the last common ancestor of cell life forms (LUCA), however, the presence of such a proton enzyme in LUCA contradicts our hypotheses about the inability of LUCA to support the proton potential on the membrane. We conducted a phylogenomic analysis of this enzyme and showed that its phylogenetic tree is better explained in terms of horizontal transfer from bacteria to archaea (Dibrova et al., 2013; Dibrova et al., 2017).

The tools developed in our laboratory are mainly of applied value. They facilitate the analysis of a large amount of data by visualizing it in a convenient form or by simplifying the performance of routine tasks.

P-loop ATPases and GTPases are widespread in all groups of living organisms; this superfamily contains about 200 different protein families, which include the catalytic subunits of rotary ATP synthases, DNA and RNA helicases, kinesins and myosins, ABC transporters, translation factors, G-proteins. About 20% of cell genes are encoding certain ATPases and GTPases. However, there is still no common understanding of the mechanism of catalysis of nucleotide triphosphate hydrolysis. We participated in a large-scale comparative structural analysis of the catalytic site of P-loop ATPases and GTPases, which involved more than 3100 structures (Kozlova et al., 2022a; Kozlova et al., 2023b). An explanation for the catalytic mechanism of these enzymes has been proposed. It includes both the process of activation by introducing positively charged residues into the active site (the so-called “lysine/arginine fingers”), and the process of activation of the water molecule for a nucleophilic attack on the γ-phosphate of the nucleotide triphosphate due to conserved residues of the Walker A and Walker B motifs (serine/threonine and aspartate), which in turn constitute an invariant in the large superfamily of P-loop-containing ATPases and GTPases.