Modern diversification of the amino acid repertoire driven by oxygen

Abstract

All extant life employs the same 20 amino acids for protein biosynthesis. Studies on the number of amino acids necessary to produce a foldable and catalytically active polypeptide have shown that a basis set of 7-13 amino acids is sufficient to build major structural elements of modern proteins. Hence, the reasons for the evolutionary selection of the current 20 amino acids out of a much larger available pool have remained elusive. Here, we have analyzed the quantum chemistry of all proteinogenic and various prebiotic amino acids. We find that the energetic HOMO-LUMO gap, a correlate of chemical reactivity, becomes incrementally closer in modern amino acids, reaching the level of specialized redox cofactors in the late amino acids tryptophan and selenocysteine. We show that the arising prediction of a higher reactivity of the more recently added amino acids is correct as regards various free radicals, particularly oxygen-derived peroxyl radicals. Moreover, we demonstrate an immediate survival benefit conferred by the enhanced redox reactivity of the modern amino acids tyrosine and tryptophan in oxidatively stressed cells. Our data indicate that in demanding building blocks with more versatile redox chemistry, biospheric molecular oxygen triggered the selective fixation of the last amino acids in the genetic code. Thus, functional rather than structural amino acid properties were decisive during the finalization of the universal genetic code.

Keywords: amino acids; genetic code; molecular oxygen; origin of life; redox reactivity.

Fig. 1.

AA HOMO–LUMO gaps. HOMO–LUMO gaps of 62 Murchison meteorite AAs, 21 genetically encoded AAs, and various metabolic descendants of the shikimate pathway (listed in Table S1) were calculated using semiempirical methods (AM1). The 21 proteinogenic AAs are plotted in the consensus order of their evolutionary appearance according to Trifonov (17), which is in agreement with many single-factor assumptions (e.g., the early introduction of prebiotic AAs, the late introduction of single-codon AAs, or the late introduction of AAs with unusual aminoacyl-tRNA synthetases). Prebiotic AAs identified in cell-free systems (5) and codon capture AAs (12) are indicated. The results of corresponding calculations using a different basis set (PM6) and an ab initio algorithm [Hartree–Fock 6–31+G(d)] are shown in Figs. S1 and S2, respectively. Individual differences notwithstanding, they recapitulate the general principle.

Fig. 2.

AA frontier orbital energies. Individual frontier orbital energies are shown of the same compounds as analyzed in Fig. 1. In this representation, small HOMO–LUMO gaps appear in the upper right edge of the graph. All 62 Murchison meteorite AAs and the first 14 genetically encoded AAs cluster in the lower left edge of the graph. Colors and abbreviations are used as in Fig. 1.

Fig. 3.

Redox reactivity of AAs. (A and B) Effect of the 20 standard AAs in a peroxyl radical scavenging assay. The relative activity of the AAs was tested at ratios 1:3 (A) and 1:2,000 (B) of AA versus radical initiator (n = 3). All AAs below a certain HOMO–LUMO threshold (∼10 eV) demonstrated activity under the conditions in A, except phenylalanine, whose inertia might be related to its unusually high radicalization enthalpy (Table S3). W and Y were the most effective AAs at high initiator/scavenger ratios (B). (C) Chemical structures of W, NAc-W-OEt, and NDo-W-OEt. (D) Inhibition of lipid peroxidation by acetylated or dodecanoylated AA ethyl esters (100 µM). Ferrous iron-induced lipid peroxidation was monitored by measuring the formation of malondialdehyde (F₁ = 379, df = 1; F₂ = 31, df = 12; ^aP < 0.001, ^bP < 0.001 versus “OXID” by post hoc test, n = 3 for compounds, n = 9 for controls) and 8-isoprostane [F₁ = 35, df = 1; F₂ = 11, df = 12; ^cP = 0.002, ^dP = 0.001 versus “OXID” by post hoc test, n = 2 for compounds, n = 4 for controls (all from duplicates)]. (E) Fluorescence microscopic images of the effect of NDo-W-OEt and NDo-F-OEt (10 µM) on the survival of neurons treated with 100 µM tert-butyl hydroperoxide (tBuOOH). Cells were immunostained for microtubule-associated protein 2 (red) indicative of neuronal survival and counterstained with the chromatin marker dye Hoechst 33342 (blue). (F) Quantification of cell survival experiments conducted as in E. Only lipophilic W and Y derivatives elicited cytoprotective activity against peroxide toxicity (F₁ = 946, df = 1; F₂ = 133, df = 9; *P < 0.001 versus “OXID” by post hoc test, n = 3 for compounds, n = 6 for controls). (G) Survival of fibroblasts treated with 50 µM tBuOOH in the presence of different AA derivatives at 10 µM concentration (F₁ = 511, df = 1; F₂ = 48, df = 12; *P < 0.001 versus “OXID” by post hoc test, n = 3 for compounds, n = 9 for controls).

Fig. 4.

Specificity of W. (A) Chemical structures of BFA and BTA. (B) W, BFA, and BTA as scavengers of peroxyl radicals under conditions as in Fig. 3B (n = 3). (C) Inhibition of lipid peroxidation by NDo-W, NDo-BFA, and NDo-BTA under conditions as in Fig. 3D [malondialdehyde: F₁ = 379, df = 1; F₂ = 31, df = 12; ^aP < 0.001, ^bP < 0.001 versus “NDo-W” by post hoc test, n = 3 for compounds, n = 9 for controls; 8-isoprostane: F₁ = 35, df = 1; F₂ = 11, df = 12; ^cP = 0.014, ^dP = 0.001 versus “NDo-W” by post hoc test, n = 2 for compounds, n = 4 for controls (all from duplicates)]. (D) Survival of tBuOOH-treated fibroblasts in the presence of NDo-W, NDo-BFA, and NDo-BTA under conditions as in Fig. 3G (F₁ = 511, df = 1; F₂ = 48, df = 12; *P < 0.001 versus “NDo-W” by post hoc test, n = 3 for compounds, n = 9 for controls).