Experimental evidence for the thermophilicity of ancestral life Satoshi Akanumaa, Yoshiki Nakajimaa, Shin-ichi Yokoboria, Mitsuo Kimuraa, Naoki Nemotoa,1, Tomoko Maseb, Ken-ichi Miyazonob, Masaru Tanokurab, and Akihiko Yamagishia,2 aDepartment of Applied Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan; and bDepartment of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Edited by David Baker, University of Washington, Seattle, WA, and approved May 29, 2013 (received for review May 1, 2013) Theoretical studies have focused on the environmental tempera- kinase (NDK) sequences as the resurrection targets. NDK cata- ture of the universal common ancestor of life with conflicting lyzes the transfer of a phosphate from a nucleoside triphosphate conclusions. Here we provide experimental support for the exis- to a nucleoside diphosphate. The ancestral NDK may have arisen tence of a thermophilic universal common ancestor. We present early, because most extant organisms contain at least one gene the thermal stabilities and catalytic efficiencies of nucleoside di- that encodes NDK. The sequences of extant NDKs are rela- phosphate kinases (NDK), designed using the information con- tively well conserved, allowing inference of the ancestral se- fi tained in predictive phylogenetic trees, that seem to represent quence with con dence. In addition, a number of 3D structures the last common ancestors of Archaea and of Bacteria. These are available for the NDK family of proteins isolated from a enzymes display extreme thermal stabilities, suggesting thermo- wide variety of organisms, including bacteria, archaea, and eukar- philic ancestries for Archaea and Bacteria. The results are robust to yotes. Therefore, NDK is an ideal model for studying the phys- ical characteristics of ancient proteins. By resurrecting ancestral the uncertainties associated with the sequence predictions and to NDK sequences and characterizing their properties, which should the tree topologies used to infer the ancestral sequences. More- reflect the ancestor’s characteristics and its environment, we over, mutagenesis experiments suggest that the universal ances- experimentally estimated the environmental temperature of tor also possessed a very thermostable NDK. Because, as we show, the Commonote that would have existed about 3,800 million the stability of an NDK is directly related to the environmental years ago (24). temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished Results and Discussion at a very high temperature. Ancestral Sequence Reconstruction Using a Small NDK Sequence Set. The first step in the reconstruction of an ancestral sequence is to ancient protein | crystal structure | last universal common ancestor | prepare a multiple amino acid sequence alignment, using the molecular resurrection | phylogenetic analysis sequences of a given protein from extant species, which then is used to build a phylogenetic tree (12). Two methods, the maxi- lucidation of the origin and early evolution of life is funda- mum-likelihood (ML) method (25) and the Bayesian method Emental to our understanding of ancient living systems and (26), have been used commonly for tree building and recon- their environment(s). One debate about the last universal com- structing the ancestral sequence. In the ML method, the likeli- mon ancestor, which has been denoted “LUCA,”“LCA,” or hood of each type of amino acid at each position in the sequence “senancestor,” and which we call “Commonote” (1), is its envi- associated with the deepest node of the tree is computed using ronmental temperature. In a well-referenced phylogenetic tree a statistical model of evolution. The ancestral sequence is defined containing small-subunit rRNA sequences, those of hyper- as the set of residues in which each residue has the greatest thermophilic archaea and bacteria are found at the deepest and likelihood of existing at its associated position. The Bayesian shortest branches (2), and therefore it has been proposed that method integrates uncertainties associated with the tree topology, the common ancestors of Archaea and Bacteria were hyper- branch lengths, and substitution models into the ancestral se- thermophilic (3, 4). Given the apparent hyperthermophilic ancestry quence calculation, whereas only the most likely estimate of the for both lineages, Occam’s razor suggests that the Commonote tree and substitution models are assumed with the ML method. was a thermophilic organism. However, although some theo- As a suboptimal test case, we first constructed an ML tree retical studies that focused on the environmental temperature from 66 extant NDK amino acid sequences (Fig. S1A). Then 10 of the Commonote support the thermophilic common-ancestry sequences representing the main tree branches were selected to hypothesis (5–7), other theoretical studies have concluded that build an ML phylogenetic tree by CODEML in PAML (27) and the universal ancestor was not (hyper)thermophilic (8–10). a Bayesian tree by nhPhybloBayes (28) to identify the deepest Therefore, the available theoretical studies disagree among them- archaeal and bacterial nodes (Fig. S1B). The CODEML-derived selves. More seriously, these theoretical studies have not been tree and the nhPhyloBayes-built tree have the same topology. tested empirically. The ancestral sequences predicted by CODEML are designated Information concerning the properties of ancient proteins is embedded in the sequences of their descendants, so the ances- tral sequence of a protein can be inferred by comparing a large Author contributions: S.A. and A.Y. designed research; S.A., Y.N., S.-i.Y., M.K., N.N., T.M., number of extant homologous sequences (11–14). A powerful and K.-i.M. performed research; S.A., Y.N., S.-i.Y., M.T., and A.Y. analyzed data; and S.A., method for experimentally studying the properties of ancient K.-i.M., M.T., and A.Y. wrote the paper. life resurrects ancestral protein sequences, thereby allowing The authors declare no conflict of interest. characterization of their features (15–23). Such resurrections This article is a PNAS Direct Submission. have been used to understand the evolution of ethanol pro- Data deposition: The crystal structures of Arc1 and Bac1 have been deposited in the duction and consumption in yeast (16), the evolutionary tra- Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank jectory of changes in ligand specificity of hormone receptors (17, (www.rcsb.org) [accession numbers 3VVT (Arc1), and 3VVU (Bac1)]. 18, 20), and the evolution of the increased complexity of vacu- 1Present address: Department of Life and Environmental Sciences, Faculty of Engineering, + olar H -ATPases (22). This empirical technique has provided Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-0016, Japan. new ways to elucidate the environmental temperatures experi- 2To whom correspondence should be addressed. E-mail: [email protected]. EVOLUTION enced by ancient bacteria (15, 19). For the study reported here, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. we chose archaeal and bacterial ancestral nucleoside diphosphate 1073/pnas.1308215110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1308215110 PNAS | July 2, 2013 | vol. 110 | no. 27 | 11067–11072 Downloaded by guest on September 24, 2021 Table 1. Tm (°C) for A. fulgidus (Afu), T. thermophilus (Tth), and Therefore, the thermal stability of the reconstructed proteins the resurrected ancestral NDKs may be an artifact of the reconstruction methods (29). Protein pH 6.0 pH 7.6 Ancestral Sequences Inferred from 204 NDK Sequences. The accuracy Archaeal of a predicted ancestral sequence depends on sequence sam- Afu NDK 100 n.d.* pling, which can be improved by including as many extant Arc1 114 113 sequences as possible in the reconstruction. Since we performed Arc2 109 109 the aforementioned experiment, improvements in computational Arc3 112 111 power have allowed the use of a larger dataset. Therefore we † Arc3sec 109 n.d. built two ML phylogenic trees from 204 extant NDK sequences. Arc4 109 110 [The Bayesian approach was not used for this tree-building ex- † ercise because Thornton and colleagues (30) recently reported Arc4sec 99 n.d. that the ML phylogenetic algorithm accurately reconstructs an- Arc5 108 107 cestral sequences.] One tree was built without constraints (Fig. Bacterial 1A and Fig. S4A), and one was built with the constraint that Tth NDK 99 99 Archaea and Bacteria each represent a monophyletic group (Fig. Bac1 99 101 1B and Fig. S4B). For both trees, the major phyla are well Bac2 98 101 grouped into their own monophyletic groups. However, the re- Bac3 109 109 † lationship among the phyla is slightly different for the two trees. Bac3sec 108 n.d. In the tree built without constraint, certain archaeal sequences Bac4 102 99 (those of Desulfurococcales and Thermoplasmatales) are para- † Bac4sec 98 n.d. phyletic and are found among the bacterial sequences, position- Bac5 107 105 ings that are inappropriate for a nearly universal phylogenetic tree. In the constrained tree, the Desulfurococcales and Thermo- Tm values were estimated from the data shown in Figs. S3 and S5B. plasmatales sequences also are paraphyletic, but they are posi- *Tm for A. fulgidus NDK could not be determined