Urzymology: Experimental Access to a Key Transition in the Appearance of Enzymes

Carter, Charles W.

doi:10.1074/jbc.r114.567495

Cited by 54 publications

(73 citation statements)

References 69 publications

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“…We recently confirmed the prediction quantitatively, by constructing and carrying out a modular thermodynamic cycle 44 . Modular cycles are enabled for TrpRS by the characterization of Urzymes 62–64 (and potentially of protozymes 26 ). As the CP1 and ABD domains are definitively deleted, the TrpRS Urzyme itself plays the role of a molecular knockout.…”

Section: Resultsmentioning

confidence: 60%

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

Carter

Chandrasekaran

Weinreb

et al. 2017

Structural Dynamics

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We measured and cross-validated the energetics of networks in Bacillus stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) using both multi-mutant and modular thermodynamic cycles. Multi-dimensional combinatorial mutagenesis showed that four side chains from this “molecular switch” move coordinately with the active-site Mg2+ ion as the active site preorganizes to stabilize the transition state for amino acid activation. A modular thermodynamic cycle consisting of full-length TrpRS, its Urzyme, and the Urzyme plus each of the two domains deleted in the Urzyme gives similar energetics. These dynamic linkages, although unlikely to stabilize the transition-state directly, consign the active-site preorganization to domain motion, assuring coupled vectorial behavior.

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Section: Resultsmentioning

confidence: 60%

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

Carter

Chandrasekaran

Weinreb

et al. 2017

Structural Dynamics

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“…Our parallel work with Class I and II Urzymes (4–9,13,14) and Protozymes (3) produced three lines of evidence that the two aaRS superfamilies evolved from opposite strands of the same gene. (i) Remarkably, and wholly unexpectedly, catalytic activity in both superfamilies arises largely from those modules that can be aligned sense/antisense, showing only second-order dependence on modules that cannot be so aligned (9).…”

Section: Introductionmentioning

confidence: 99%

An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures

Sapienza

Williams

et al. 2016

ACS Chem. Biol.

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Urzymes—short, active core modules derived from enzyme superfamilies—prepared from the two aminoacyl-tRNA synthetase (aaRS) Classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of 15N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of 1H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of alpha helix and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term “molten globule” is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ~60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.

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“…The fidelity with which the acceptor stem code might have been interpreted is now accessible to experiment because the modular componentsaaRS urzymes and tRNA acceptor stem helices-have now both been characterized (13-16, 41, 42). Preliminary specificity ratios [(k cat /K M ) (cognate) /(k cat /K M ) (noncognate) ] for activation of cognate vs. all 19 noncognate amino acids by LeuRS and HisRS urzymes (13,15,36,43) suggest that they prefer amino acids from their own class by ∼−1 kcal/mol, or ∼20% of −5.5 kcal/mol based on the present day specificity ratio of 10 −4 (44). In due course, k cat /K M values for aminoacylation of different tRNAs by urzymes will presumably be determined.…”

Section: Independent Trna Coding Strategies Suggest Distinct Stages Ofmentioning

confidence: 99%

tRNA acceptor stem and anticodon bases form independent codes related to protein folding

Carter

Wolfenden

2015

Proc. Natl. Acad. Sci. U.S.A.

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Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3′ acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each sidechain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for β-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or β-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water.genetic code | aminoacyl-tRNA synthetases | urzymes | multivariate regression | protein folding T he genetic code is implemented by two distinct superfamilies of protein-RNA complexes between an aminoacyl-tRNA synthetase (aaRS) from one of two classes (1, 2) and its cognate tRNA. These recognition complexes effect the transfer of activated amino acids to the correct tRNA molecule, producing aminoacyl-tRNAs needed for protein synthesis by the ribosome. Errors in charging are rare (3, 4), and it is generally agreed that the low frequency of mischarging is based on synthetase recognition of specific identity elements in tRNA molecules (5). Many investigators (6-8) have observed that the codon table tends to reduce deleterious effects of point mutations (9) by assuring that they do minimal violence to the physical requirements of protein folding. One earlier study (10) identified a nonrandom tendency for hydrophilic side-chains to be coded by an A as the second codon base, hinting at more extensive relationships between the code and factors that direct protein folding.tRNA identity elements (5, 11) map to both the anticodon and acceptor stem at opposite ends of the L-shaped tRNA molecule and are distinct from binding determinants for elongation factorTu in the T-stem (12). Invariant cores of both classes of aaRS, termed urzymes (from the prefix ur-= primitive), lack anticodon-binding domains and cannot recognize the anticodon. However, they catalyze amino acid activation and acyl transfer with K M values approaching those of contemporary aaRS, consistent with their participation in early protein synthesis (13-16). The implied ability of ancestral aaRS to recognize tRNA acceptor stems, but not anticodons, is consistent with the suggestion that the earliest proteins were coded no...

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Urzymology: Experimental Access to a Key Transition in the Appearance of Enzymes

Cited by 54 publications

References 69 publications

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures

tRNA acceptor stem and anticodon bases form independent codes related to protein folding

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