The structure of a truncated phosphoribosylanthranilate isomerase suggests a unified model for evolution of the (βα)8 barrel fold

The (βα)₈ barrel is one of the most common protein folds, and enzymes with this architecture display a remarkable range of catalytic activities. Many of these functions are associated with ancient metabolic pathways, and phylogenetic reconstructions suggest that the (βα)₈ barrel was one of the very first protein folds to emerge. Consequently, there is considerable interest in understanding the evolutionary processes that gave rise to this fold. In particular, much attention has been focused on the plausibility of (βα)₈ barrel evolution from homodimers of half barrels. However, we previously isolated a three-quarter-barrel-sized fragment of a (βα)₈ barrel, termed truncated phosphoribosylanthranilate isomerase (trPRAI), that is soluble and almost as thermostable as full-length N-(5′-phosphoribosyl)anthranilate isomerase (PRAI). Here, we report the NMR-derived structure of trPRAI. The subdomain is monomeric, is well ordered and adopts a native-like structure in solution. Side chains from strands β₁ (Glu3 and Lys5), β₂ (Tyr25) and β₆ (Lys122) of trPRAI repack to shield the hydrophobic core from the solvent. This result demonstrates that three-quarter barrels were viable intermediates in the evolution of the (βα)₈ barrel fold. We propose a unified model for (βα)₈ barrel evolution that combines our data, previously published work and plausible scenarios for the emergence of (initially error-prone) genetic systems. In this model, the earliest proto-cells contained diverse pools of part-barrel subdomains. Combinatorial assembly of these subdomains gave rise to many distinct lineages of (βα)₈ barrel proteins, that is, our model excludes the possibility that there was a single (βα)₈ barrel from which all present examples are descended.     

Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.     

A study in molecular contingency: glutamine phosphoribosylpyrophosphate amidotransferase is a promiscuous and evolvable phosphoribosylanthranilate isomerase

The prevalence of paralogous enzymes implies that novel catalytic functions can evolve on preexisting protein scaffolds. The weak secondary activities of proteins, which reflect catalytic promiscuity and substrate ambiguity, are plausible starting points for this evolutionary process. In this study, we observed the emergence of a new enzyme from the ASKA (A Complete Set of E. coli K-12 ORF Archive) collection of Escherichia coli open reading frames. The overexpression of (His)₆-tagged glutamine phosphoribosylpyrophosphate amidotransferase (PurF) unexpectedly rescued a ΔtrpF E. coli strain from starvation on minimal media. The wild-type PurF and TrpF enzymes are unrelated in sequence, tertiary structure and catalytic mechanism. The promiscuous phosphoribosylanthranilate isomerase activity of the ASKA PurF variant apparently stems from a preexisting affinity for phosphoribosylated substrates. The relative fitness of the (His)₆-PurF/ΔtrpF strain was improved 4.8-fold to nearly wild-type levels by random mutagenesis of purF and genetic selection. The evolved and ancestral PurF proteins were purified and reacted with phosphoribosylanthranilate in vitro. The best evolvant (k_cat/K_M = 0.3 s^− 1 M^− 1) was ∼ 25-fold more efficient than its ancestor but > 10⁷-fold less efficient than the wild-type phosphoribosylanthranilate isomerase. These observations demonstrate in quantitative terms that the weak secondary activities of promiscuous enzymes can dramatically improve the fitness of contemporary organisms.     

Protein Design through Systematic Catalytic Loop Exchange in the (β/α)8 Fold

Protein engineering by directed evolution has proven effective in achieving various functional modifications, but the well-established protocols for the introduction of variability, typically limited to random point mutations, seriously restrict the scope of the approach. In an attempt to overcome this limitation, we sought to explore variant libraries with richer diversity at regions recognized as functionally important through an exchange of natural components, thus combining design with combinatorial diversity. With this approach, we expected to maintain interactions important for protein stability while directing the introduction of variability to areas important for catalysis.Our strategy consisted in loop exchange over a (β/α)₈ fold. Phosphoribosylanthranilate isomerase was chosen as scaffold, and we investigated its tolerance to loop exchange by fusing variant libraries to the chloramphenicol acetyl transferase coding gene as an in vivo folding reporter. We replaced loops 2, 4, and 6 of phosphoribosylanthranilate isomerase with loops of varied types and sizes from enzymes sharing the same fold.To allow for a better structural fit, saturation mutagenesis was adopted at two amino acid positions preceding the exchanged loop. Our results showed that 30% to 90% of the generated mutants in the different libraries were folded. Some variants were selected for further characterization after removal of chloramphenicol acetyl transferase gene, and their stability was studied by circular dichroism and fluorescence spectroscopy. The sequences of 545 clones show that the introduction of variability at “hinges” connecting the loops with the scaffold exhibited a noticeable effect on the appearance of folded proteins. Also, we observed that each position accepted foreign loops of different sizes and sequences.We believe our work provides the basis of a general method of exchanging variably sized loops within the (β/α)₈ fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity.