Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Abstract

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.

Keywords: ATP; Rodin-Ohno hypothesis; amino acid activation; aminoacyl tRNA synthetase; chemical biology; enzyme catalysis; gene structure; origin of life; protein design; sense/antisense genetic coding.

FIGURE 1.

Deconstruction of Class I tryptophanyl (PDB code 1MAU)- and II histidyl (PDB code 2EL9)-tRNA synthetases into successively smaller fragments that retain catalytic activity (1, 7–10, 43). Graphics for smaller constructs are derived from coordinates of the full-length enzymes. Colored bars below each structure denote the modules contained within each structure; white segments are deleted. The number of amino acids (aa) in each construct is noted. Measured catalytic rate enhancements relative to the uncatalyzed second-order rate (k_cat/K_m)/k_non are plotted on vertical scales aligned in the center of the figure and are colored from blue (slower) to red (faster). The 46-mers are the subject of this work.

FIGURE 2.

Sense/antisense gene coding for Class I and II ATP-binding sites from opposite strands. A, schematic of gene architecture. ATP and amino acid-binding sites of the two antisense gene products reflect across the nucleic acid backbone symmetry axis (center), so that ATP-binding sites are at the N terminus of Class I and C terminus of Class II aaRSs. Structural schematics drawn from PDB files output by Rosetta for Class I (left) Class II (right) 46-mers to suggest possible folded configurations are in similar but not identical orientations to those in Fig. 1. These configurations reflect structural constraints input to Rosetta. Spheres are activated aminoacyl (magenta)-5′-AMP (yellow). B, sequences designed by Rosetta to stabilize tertiary structures in the top panel, preserving coding sequence complementarity. The inset shows the Class I catalytic HIGH and Class II motif 2 signatures; nucleic acid sequence of the gene is shown in Fig. 6A. Red letters denote mutations made to catalytic residues.

FIGURE 3.

Gel showing expression and purification of designed and active site mutants of the Class I (I-mer) and Class II (II-mer). MBP is maltose-binding protein; Lys is crude lysate; sol is soluble supernatant after lysis; and elute is the fraction eluted from nickel-NTA and assayed.

FIGURE 4.

Fluorescence changes due to the presence of ATP, derived from titrations of ATP with TrpRS (A and C) and HisRS (B and D) 46-mer peptides. Integrated (300–450 nm) spectra for peptides without ATP (A and B, black) were subtracted from those with ATP (A and B, gray) to estimate quenching of ATP fluorescence by the peptides and of the peptides by ATP. ATP-dependent fluorescence changes were fitted to Equation 1 for Trp-46-mer (C) and His-46-mer (D). Dashed lines indicate data points omitted from fitting because of development of turbidity developing at high [peptide]. Fitted dissociation constants are given in the text.

FIGURE 5.

Steady-state kinetic parameters for the WT TrpRS and HisRS 46-mer peptides. Assays were conducted as described under “Materials and Methods.” Data points to the left of vertical lines at 100 mm along the x axis cannot be interpreted unambiguously, because the substrate concentrations are below the catalyst concentration. Velocities are therefore underestimates.

FIGURE 6.

Sense/antisense gene. A, DNA sequence, with codon/anticodon pairs translated. B, comparison of the sense antisense gene designed by Rosetta (30) (center) with Logos derived from multiple alignments for ∼200 sequences for each of six contemporary Class IA, -B, and -C and five Class IIA, -B, and -C sequences (Fig. prepared using Canvas 8.0 and WebLogo (55)). Rosetta was not allowed to vary the five blue amino acids in the designed sequence. Ovals are positions where, elsewhere, Rosetta chose consensus amino acids from the biological MSA. Percentages to the right indicate a wide variance in such selection between the Class I and II 46-mers as discussed in the text.

FIGURE 7.

Distributions of [³²P]ATP produced per peptide for all assays for designed 46-residue sense/antisense Class I and II peptide catalysts, MBP controls, and mutant peptides. Distributions of [³²P]ATP produced per peptide for all assays for the designed 46-residue sense/antisense Class I and II peptide catalysts, MBP controls, and mutant peptides are shown. All data were divided by concentration. White arrows denote averages of the distributions, and vertical cross-bars at the tails of the arrows give their standard errors of the mean. The y axis corresponds to the ³²PP_i exchange data, in units of mole of ATP/mol of peptide. Left, MBP controls. Center, distributions for designed peptides. Right, mutant peptides. Differences between peptide and MBP controls have Z-scores of 10.4 and 9.8. The differences for Class I and II peptides have Z-scores of 3.0 and 11.4. Prepared using JMP (38).

FIGURE 8.

Time, class, and peptide concentration (0. 44 μm; 1.75 μm) dependences of catalytic peptide activity. The y axis is in [³²P]ATP concentration. Variances of the time dependences are indicated by shading along either side of the fitted lines. Prepared using JMP (35).

FIGURE 9.

Estimation of apparent second-order rate constants. Amino acid concentration dependences plotted in Michaelis-Menten format. A–D, data for all time points for each unique combination of peptide and substrate concentrations were fitted against time in seconds by linear least squares. The mean values of rates for both amino acid concentrations were plotted against amino acid concentration for experiments with 0.44 μm (A and B) and 1.75 μm peptide (C and D), giving two independent experimental estimates of steady-state kinetic parameters for Class I (A and C) and Class II (B and D) peptides, which are given in Table 4.

FIGURE 10.

Significant clustering of steady-state parameters for wild-type (amber) and designed (blue) Class I and II 46-mer peptides. Plots show observed values for the free energies of activation derived from k_cat and binding, K_m, versus calculated values from linear regression models using the coefficients for the binary descriptors: Designed {0,1}, Class {1(▴),2(●} (k_cat only), and substrate {ATP (*) = 1, amino acid (●) = 2} (K_m only). A, two designed 46-mers have substantially higher turnover numbers (〈ΔGk_cat〉 = 5.8 kcal/mol) than the wild-type 46-mers (〈ΔGk_cat〉 = 9.2 kcal/mol), and the TrpRS (Class I) wild-type 46-mer has significantly higher turnover numbers than the HisRS (Class II) 46-mer with both amino acid and ATP substrates. B, both wild-type 46-mers bind amino acid substrates with higher affinities (〈ΔGK_M〉 = 5.9 kcal/mol) than does either designed 46-mer (〈ΔGK_M〉 = 2.7 kcal/mol). Similarly, wild-type 46-mers have higher affinity for ATP than they do for amino acid substrates. Student's t test probabilities for these effects under the null hypothesis are <0.0001 for WT sequences in both models, 0.01 for Class in the model for ΔGk_cat, and 0.01 for substrate in the model for ΔGK_M, so these differences are statistically significant.