Urzymology: experimental access to a key transition in the appearance of enzymes

Abstract

Urzymes are catalysts derived from invariant cores of protein superfamilies. Urzymes from both aminoacyl-tRNA synthetase classes possess sophisticated catalytic mechanisms: pre-steady state bursts, significant transition-state stabilization of both amino acid activation, and tRNA acylation. However, they have insufficient specificity to ensure a fully developed genetic code, suggesting that they participated in synthesizing statistical proteins. They represent a robust experimental platform from which to articulate and test hypotheses both about their own ancestors and about how they, in turn, evolved into modern enzymes. They help reshape numerous paradigms from the RNA World hypothesis to protein structure databases and allostery.

Keywords: Aminoacyl tRNA Synthetase; Bioinformatics; Genetic Code; Precellular Evolution; Protein Engineering; Protein Evolution; Protein Synthesis.

FIGURE 1.

Urzymology and molecular evolution. A, proposed role of aaRS Urzymes in the development of codon-dependent translation (yellow background). The scale indicates time in years since the earth formed. Urzymes are rehabilitated forms of invariant structural cores of modern enzyme superfamilies. Many modern enzymes likely preceded the last universal common ancestor and first “organism” (LUCA). B, methods appropriate for studying objects and processes along the timeline in A. Because urzymology connects the earliest genetic coding to the emergence of modern enzymes, it affords a powerful enabling technology for studying key transitions in the evolution of the genetic code.

FIGURE 2.

Minimal Glutaminyl-tRNA synthetase (20) and its putative Urzyme (firebrick ribbon). Selection of functional deletions from CP1 (100 amino acids; blue) and the ABD (134 amino acids; green) yielded a minimal enzyme (wheat) that is still twice the size of the invariant core used to construct TrpRS and LeuRS Urzymes. Active-site ligands are shown as spheres.

FIGURE 3.

Quantitative framework for assessing the catalytic significance of Urzymes and other putative stages of aaRS evolution. A, rate accelerations estimated from experimental data for single (red) and bisubstrate (black and bold) reactions adapted from Ref. to include uncatalyzed and catalyzed rates of bisubstrate reactions of the ribosome (71), amino acid activation (18), and kinases (72). Abbreviations: CAN, carbonic anhydrase; CMU, chorismate mutase; KSI, ketosteroid isomerase; RIBO, ribosome; CDA, cytidine deaminase; PEP, carboxypeptidase B; MAN, mandelate racemase; KINA, hexokinase; FUM, fumarase; GLU, β-glucosidase; ODC, orotidylate carboxylase; ADC, amino acid decarboxylase; IPT, inositol phosphatase. Second order rate constants (black bars) were converted into comparable units by multiplying by 0.002 m, which is the ATP concentration used to assay the catalysts shown in B. B, experimental rate accelerations estimated from steady state kinetics as k_cat/K_m for a series of catalysts derived from Class I and Class II aaRS. A and B have the same vertical scales, and the origin in B has been set equal to the uncatalyzed rate of amino acid activation (AAact) in A. Red bars, Class I TrpRS and LeuRS (see Footnote 3) constructs; blue bars, Class II HisRS constructs; green bar, a ribozymal catalyst (38), for comparison. Research presented in A and B was originally published in Ref. . © The American Society for Biochemistry and Molecular Biology. C, Class I LeuRS and Class II HisRS Urzyme amino acid specificities. More negative ΔGk_cat/K_m values indicate higher activities. By ∼1 kcal/mole (light bands) or ∼5-fold, each Urzyme prefers substrates from its own class (dark bands).

FIGURE 4.

Structural homology between Class I Urzymes and the TOPRIM domain found in topoisomerases and primases (41). Core domain structures are superimposed in the center. Insertion points corresponding to Class I CP1 and CP2 (42) are shared by various TOPRIM domains, as indicated. The N and C termini of the insertions are indicated by red spheres and correspond closely throughout the domain superfamily. P. horikoshii, Pyrococcus horikoshii; D. radiodurans, Deinococcus radiodurans; E. coli, Escherichia coli.