Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.
References
[1]
Khersonsky O, Tawfik DS. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem. 2010;79:471–505. doi: 10.1146/annurev-biochem-030409-143718. pmid:20235827
[2]
Voordeckers K, Brown CA, Vanneste K, van der Zande E, Voet A, Maere S, et al. Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication. PLoS Biol. 2012;10(12):e1001446. doi: 10.1371/journal.pbio.1001446. pmid:23239941
[3]
Gould SJ, Vrba ES. Exaptation-a missing term in the science of form. Paleobiology. 1982;8(1):4–15.
[4]
Tomarev SI, Piatigorsky J. Lens crystallins of invertebrates-diversity and recruitment from detoxification enzymes and novel proteins. Eur J Biochem. 1996;235(3):449–65. pmid:8654388 doi: 10.1111/j.1432-1033.1996.00449.x
[5]
Barve A, Wagner A. A latent capacity for evolutionary innovation through exaptation in metabolic systems. Nature. 2013;500(7461):203–6. doi: 10.1038/nature12301. pmid:23851393
[6]
Henn-Sax M, Thoma R, Schmidt S, Hennig M, Kirschner K, Sterner R. Two (βα)8-barrel enzymes of histidine and tryptophan biosynthesis have similar reaction mechanisms and common strategies for protecting their labile substrates. Biochemistry. 2002;41(40):12032–42. pmid:12356303 doi: 10.1021/bi026092h
[7]
Mirkin BG, Fenner TI, Galperin MY, Koonin EV. Algorithms for computing parsimonious evolutionary scenarios for genome evolution, the last universal common ancestor and dominance of horizontal gene transfer in the evolution of prokaryotes. BMC Evol Biol. 2003;3(1):2.
[8]
Barona-Gómez F, Hodgson DA. Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis. EMBO Rep. 2003;4(3):296–300. pmid:12634849 doi: 10.1038/sj.embor.embor771
[9]
Due AV, Kuper J, Geerlof A, von Kries JP, Wilmanns M. Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis. Proc Natl Acad Sci U S A. 2011;108(9):3554–9. doi: 10.1073/pnas.1015996108. pmid:21321225
[10]
Kuper J, D？nges C, Wilmanns M. Two-fold repeated (βα)4 half-barrels may provide a molecular tool for dual substrate specificity. EMBO Rep. 2005;6:134–9. pmid:15654319 doi: 10.1038/sj.embor.7400330
[11]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402. pmid:9254694 doi: 10.1093/nar/25.17.3389
[12]
S？derholm A, Guo X, Newton MS, Evans GB, N？svall J, Patrick WM, et al. Two-step ligand binding in a (βα)8 barrel enzyme: SUBSTRATE-BOUND STRUCTURES SHED NEW LIGHT ON THE CATALYTIC CYCLE OF HisA. J Biol Chem. 2015;290(41):24657–68. doi: 10.1074/jbc.M115.678086. pmid:26294764
[13]
Jürgens C, Strom A, Wegener D, Hettwer S, Wilmanns M, Sterner R. Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways. Proc Natl Acad Sci U S A. 2000;97(18):9925–30. pmid:10944186 doi: 10.1073/pnas.160255397
[14]
Boussau B, Blanquart S, Necsulea A, Lartillot N, Gouy M. Parallel adaptations to high temperatures in the Archaean eon. Nature. 2008;456(7224):942–5. doi: 10.1038/nature07393. pmid:19037246
[15]
Lartillot N, Lepage T, Blanquart S. PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics. 2009;25(17):2286–8. doi: 10.1093/bioinformatics/btp368. pmid:19535536
[16]
Ashkenazy H, Penn O, Doron-Faigenboim A, Cohen O, Cannarozzi G, Zomer O, et al. FastML: a web server for probabilistic reconstruction of ancestral sequences. Nucleic Acids Res. 2012;40(Web Server issue):W580–4. doi: 10.1093/nar/gks498. pmid:22661579
[17]
Noda-García L, Juárez-Vázquez AL, ávila-Arcos MC, Verduzco-Castro EA, Montero-Morán G, Gaytán P, et al. Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)8 isomerase evolutionary intermediates from a diverse metagenome. BMC Evol Biol. 2015;15:107. doi: 10.1186/s12862-015-0378-1. pmid:26058375
[18]
Reisinger B, Sperl J, Holinski A, Schmid V, Rajendran C, Carstensen L, et al. Evidence for the existence of elaborate enzyme complexes in the Paleoarchean era. J Am Chem Soc. 2014;136(1):122–9. doi: 10.1021/ja4115677. pmid:24364418
[19]
Perez-Jimenez R, Inglés-Prieto A, Zhao ZM, Sanchez-Romero I, Alegre-Cebollada J, Kosuri P, et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat Struct Mol Biol. 2011;18(5):592–6. doi: 10.1038/nsmb.2020. pmid:21460845
[20]
Patrick WM, Matsumura I. A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase. J Mol Biol. 2008;377(2):323–36. doi: 10.1016/j.jmb.2008.01.043. pmid:18272177
[21]
N？svall J, Sun L, Roth JR, Andersson DI. Real-time evolution of new genes by innovation, amplification, and divergence. Science. 2012;338(6105):384–7. doi: 10.1126/science.1226521. pmid:23087246
[22]
Noda-García L, Camacho-Zarco AR, Medina-Ruíz S, Gaytán P, Carrillo-Tripp M, Fül？p V, et al. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer. Mol Biol Evol. 2013;30(9):2024–34. doi: 10.1093/molbev/mst115. pmid:23800623
[23]
Patrick WM, Quandt EM, Swartzlander DB, Matsumura I. Multicopy suppression underpins metabolic evolvability. Mol Biol Evol. 2007;24(12):2716–22. pmid:17884825 doi: 10.1093/molbev/msm204
[24]
Hunter S, Jones P, Mitchell A, Apweiler R, Attwood TK, Bateman A, et al. InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Res. 2012;40(Database issue):D306–12. doi: 10.1093/nar/gkr948. pmid:22096229
[25]
Atkinson HJ, Morris JH, Ferrin TE, Babbitt PC. Using sequence similarity networks for visualization of relationships across diverse protein superfamilies. PLoS One. 2009;4(2):e4345. doi: 10.1371/journal.pone.0004345. pmid:19190775
[26]
Gerlt JA, Bouvier JT, Davidson DB, Imker HJ, Sadkhin B, Slater DR, et al. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for generating protein sequence similarity networks. Biochim Biophys Acta. 2015;1854(8):1019–37. doi: 10.1016/j.bbapap.2015.04.015. pmid:25900361
[27]
Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006;22(13):1658–9. pmid:16731699 doi: 10.1093/bioinformatics/btl158
[28]
Pürzer A, Grassmann F, Birzer D, Merkl R. Key2Ann: a tool to process sequence sets by replacing database identifiers with a human-readable annotation. J Integr Bioinform. 2011;8(1):153.
[29]
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504. pmid:14597658 doi: 10.1101/gr.1239303
[30]
Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011;27(3):431–2. doi: 10.1093/bioinformatics/btq675. pmid:21149340
[31]
Plach MG, L？ffler P, Merkl R, Sterner R. Conversion of anthranilate synthase into isochorismate synthase: implications for the evolution of chorismate-utilizing enzymes. Angewandte Chemie. 2015;54(38):11270–4. doi: 10.1002/anie.201505063. pmid:26352034
[32]
Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. doi: 10.1093/molbev/mst010. pmid:23329690
[33]
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17(4):540–52. pmid:10742046 doi: 10.1093/oxfordjournals.molbev.a026334
[34]
Claren J, Malisi C, H？cker B, Sterner R. Establishing wild-type levels of catalytic activity on natural and artificial (βα)8-barrel protein scaffolds. Proc Natl Acad Sci U S A. 2009;106(10):3704–9. doi: 10.1073/pnas.0810342106. pmid:19237570
[35]
Thoma R, Obmolova G, Lang DA, Schwander M, Jeno P, Sterner R, et al. Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis. FEBS Lett. 1999;454(1–2):1–6. pmid:10413084 doi: 10.1016/s0014-5793(99)00757-7
[36]
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989;77(1):51–9. pmid:2744487 doi: 10.1016/0378-1119(89)90358-2
[37]
Hommel U, Eberhard M, Kirschner K. Phosphoribosyl anthranilate isomerase catalyzes a reversible Amadori reaction. Biochemistry. 1995;34(16):5429–39. pmid:7727401 doi: 10.1021/bi00016a014
[38]
Eberhard M. A set of programs for analysis of kinetic and equilibrium data. Comput Appl Biosci. 1990;6(3):213–21. pmid:2207745 doi: 10.1093/bioinformatics/6.3.213
[39]
Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97(12):6640–5. pmid:10829079 doi: 10.1073/pnas.120163297
[40]
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A. 2000;97(11):5978–83. pmid:10811905 doi: 10.1073/pnas.100127597
[41]
Sterner R, Dahm A, Darimont B, Ivens A, Liebl W, Kirschner K. (βα)8-barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima. EMBO J. 1995;14(18):4395–402. pmid:7556082
