The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

Department of Biochemistry and Molecular Biology Center for Computational Biology and Bioinformatics Indiana University School of Medicine Indianapolis Indiana USA

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Department of Cell Biology Faculty of Science Charles University BIOCEV Prague Czech Republic

Institute of Organic Chemistry and Biochemistry IOCB Research Centre and Gilead Sciences Academy of Sciences of the Czech Republic Prague Czech Republic

Proteomics Core Facility BIOCEV Faculty of Science Charles University Prague Czech Republic

Zobrazit více v PubMed

Cleaves HJ II. The origin of the biologically coded amino acids. J Theor Biol. 2010;263:490–498. PubMed

Philip GK, Freeland SJ. Did evolution select a nonrandom "alphabet" of amino acids? Astrobiology. 2011;11:235–240. PubMed

Ilardo M, Meringer M, Freeland SJ, Rasulev B, Cleaves HJ II. Extraordinarily adaptive properties of the genetically encoded amino acids. Sci Rep. 2015;5:9414. PubMed PMC

Ilardo M, Bose R, Meringer M, et al. Adaptive properties of the genetically encoded amino acid alphabet are inherited from its subsets. Sci Rep. 2019;9:12468. PubMed PMC

Tretyachenko V, Vymětal J, Bednárová L, et al. Random protein sequences can form defined secondary structures and are well‐tolerated in vivo . Sci Rep. 2017;7:15449. PubMed PMC

Tanaka J, Doi N, Takashima H, Yanagawa H. Comparative characterization of random‐sequence proteins consisting of 5, 12, and 20 kinds of amino acids. Protein Sci. 2010;19:786–795. PubMed PMC

Newton MS, Morrone DJ, Lee KH, Seelig B. Genetic code evolution investigated through the synthesis and characterisation of proteins from reduced‐alphabet libraries. Chembiochem. 2019;20:846–856. PubMed

Higgs PG, Pudritz RE. A thermodynamic basis for prebiotic amino acid synthesis and the nature of the first genetic code. Astrobiology. 2009;9:483–490. PubMed

Trifonov EN. Consensus temporal order of amino acids and evolution of the triplet code. Gene. 2000;261:139–151. PubMed

Granold M, Hajieva P, Toşa MI, Irimie FD, Moosmann B. Modern diversification of the amino acid repertoire driven by oxygen. Proc Natl Acad Sci USA. 2018;115:41–46. PubMed PMC

Fournier GP, Alm EJ. Ancestral reconstruction of a pre‐LUCA aminoacyl‐tRNA synthetase ancestor supports the late addition of Trp to the genetic code. J Mol Evol. 2015;80:171–185. PubMed

Yang XL, Otero FJ, Skene RJ, McRee DE, Schimmel P, Ribas de Pouplana L. Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains. Proc Natl Acad Sci USA. 2003;100:15376–15380. PubMed PMC

Riddle DS, Santiago JV, Bray‐Hall ST, et al. Functional rapidly folding proteins from simplified amino acid sequences. Nat Struct Biol. 1997;4:805–809. PubMed

Akanuma S, Kigawa T, Yokoyama S. Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set. Proc Natl Acad Sci USA. 2002;99:13549–13553. PubMed PMC

Longo LM, Lee J, Blaber M. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein. Proc Natl Acad Sci USA. 2013;110:2135–2139. PubMed PMC

Shibue R, Sasamoto T, Shimada M, Zhang B, Yamagishi A, Akanuma S. Comprehensive reduction of amino acid set in a protein suggests the importance of prebiotic amino acids for stable proteins. Sci Rep. 2018;8:1227. PubMed PMC

Solis AD. Reduced alphabet of prebiotic amino acids optimally encodes the conformational space of diverse extant protein folds. BMC Evol Biol. 2019;19:158. PubMed PMC

Kimura M, Akanuma S. Reconstruction and characterization of thermally stable and catalytically active proteins comprising an alphabet of ~13 amino acids. J Mol Evol. 2020;88:372–381. PubMed

Campen A, Williams RM, Brown CJ, Meng J, Uversky VN, Dunker AK. TOP‐IDP‐scale: A new amino acid scale measuring propensity for intrinsic disorder. Protein Pept Lett. 2008;15:956–963. PubMed PMC

Burley SK, Petsko GA. Aromatic‐aromatic interaction: A mechanism of protein structure stabilization. Science. 1985;229:23–28. PubMed

Di Mauro E, Dunker AK, Trifonov EN. Disorder to order, nonlife to life: In the beginning there was a mistake. In: Seckbach J, editor. Genesis—In the beginning. New York: Springer, 2012; p. 415–435.

Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK. Sequence complexity of disordered protein. Proteins. 2001;42:38–48. PubMed

Oldfield CJ, Dunker AK. Intrinsically disordered proteins and intrinsically disordered protein regions. Annu Rev Biochem. 2014;83:553–584. PubMed

Bukhari SA, Caetano‐Anollés G. Origin and evolution of protein fold designs inferred from phylogenomic analysis of CATH domain structures in proteomes. PLoS Comput Biol. 2013;9:e1003009. PubMed PMC

Peng K, Radivojac P, Vucetic S, Dunker AK, Obradovic Z. Length‐dependent prediction of protein intrinsic disorder. BMC Bioinform. 2006;7:208. PubMed PMC

Brooks DJ, Fresco JR. Increased frequency of cysteine, tyrosine, and phenylalanine residues since the last universal ancestor. Mol Cell Proteomics. 2002;1:125–131. PubMed

Sumbalova L, Stourac J, Martinek T, Bednar D, Damborsky J. HotSpot wizard 3.0: Web server for automated design of mutations and smart libraries based on sequence input information. Nucleic Acids Res. 2018;46:W356–W362. PubMed PMC

Nurkanto A, Jeelani G, Yamamoto T, et al. Biochemical, metabolomic, and genetic analyses of dephospho coenzyme a kinase involved in coenzyme a biosynthesis in the human enteric parasite Entamoeba histolytica . Front Microbiol. 2018;9:2902. PubMed PMC

Seto A, Murayama K, Toyama M, et al. ATP‐induced structural change of dephosphocoenzyme a kinase from Thermus thermophilus HB8. Proteins. 2005;58:235–242. PubMed

Semisotnov GV, Rodionova NA, Kutyshenko VP, Ebert B, Blanck J, Ptitsyn OB. Sequential mechanism of refolding of carbonic anhydrase B. FEBS Lett. 1987;224:9–13. PubMed

Walia G, Surolia A. Insights into the regulatory characteristics of the mycobacterial dephosphocoenzyme a kinase: Implications for the universal CoA biosynthesis pathway. PLoS One. 2011;6:e21390. PubMed PMC

Alva V, Söding J, Lupas AN. A vocabulary of ancient peptides at the origin of folded proteins. Elife. 2015;4:e09410. PubMed PMC

Longo LM, Jabłońska J, Vyas P, et al. On the emergence of P‐loop NTPase and Rossmann enzymes from a Beta‐alpha‐Beta ancestral fragment. Elife. 2020;9:e64415. PubMed PMC

Romero Romero ML, Yang F, Lin YR, et al. Simple yet functional phosphate‐loop proteins. Proc Natl Acad Sci USA. 2018;115:E11943–E11950. PubMed PMC

Vyas P, Trofimyuk O, Longo LM, Deshmukh FK, Sharon M, Tawfik DS. Helicase‐like functions in phosphate loop containing beta‐alpha polypeptides. BioRxiv . 10.1101/2020.07.30.228619 PubMed DOI PMC

Vamvaca K, Vögeli B, Kast P, Pervushin K, Hilvert D. An enzymatic molten globule: Efficient coupling of folding and catalysis. Proc Natl Acad Sci USA. 2004;101:12860–12864. PubMed PMC

Pervushin K, Vamvaca K, Vögeli B, Hilvert D. Structure and dynamics of a molten globular enzyme. Nat Struct Mol Biol. 2007;14:1202–1206. PubMed

Walter KU, Vamvaca K, Hilvert D. An active enzyme constructed from a 9‐amino acid alphabet. J Biol Chem. 2005;280:37742–37746. PubMed

Sapienza PJ, Li L, Williams T, Lee AL, Carter CW Jr. An ancestral tryptophanyl‐tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground‐state tertiary structures. ACS Chem Biol. 2016;11:1661–1668. PubMed PMC

Bonfio C, Valer L, Scintilla S, et al. UV‐light‐driven prebiotic synthesis of iron‐sulfur clusters. Nat Chem. 2017;9:1229–1234. PubMed PMC

Weber AL, Pizzarello S. The peptide‐catalyzed stereospecific synthesis of tetroses: A possible model for prebiotic molecular evolution. Proc Natl Acad Sci USA. 2006;103:12713–12717. PubMed PMC

Longo LM, Tenorio CA, Kumru OS, Middaugh CR, Blaber M. A single aromatic core mutation converts a designed "primitive" protein from halophile to mesophile folding. Protein Sci. 2015;24:27–37. PubMed PMC

Longo LM, Despotović D, Weil‐Ktorza O, et al. Primordial emergence of a nucleic acid‐binding protein via phase separation and statistical ornithine‐to‐arginine conversion. Proc Natl Acad Sci USA. 2020;117:15731–15739. PubMed PMC

Longo LM, Blaber M. Protein design at the interface of the pre‐biotic and biotic worlds. Arch Biochem Biophys. 2012;526:16–21. PubMed

Longo LM, Blaber M. Prebiotic protein design supports a halophile origin of foldable proteins. Front Microbiol. 2014;4:418. PubMed PMC

Huang F, Oldfield C, Meng J, et al. Subclassifying disordered proteins by the CH‐CDF plot method. Pac Symp Biocomput. 2012;17:128–139. PubMed

Sreerama N, Woody, RW . Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem. 2000;287:252–260. PubMed

Peptides En Route from Prebiotic to Biotic Catalysis

Modern and prebiotic amino acids support distinct structural profiles in proteins

In Vitro Evolution Reveals Noncationic Protein-RNA Interaction Mediated by Metal Ions

Peptides before and during the nucleotide world: an origins story emphasizing cooperation between proteins and nucleic acids