Deciphering the Allosterically Driven Conformational Ensemble in Tryptophan Synthase Evolution

Abstract

Multimeric enzyme complexes are ubiquitous in nature and catalyze a broad range of useful biological transformations. They are often characterized by a tight allosteric coupling between subunits, making them highly inefficient when isolated. A good example is Tryptophan synthase (TrpS), an allosteric heterodimeric enzyme in the form of an αββα complex that catalyzes the biosynthesis of L-tryptophan. In this study, we decipher the allosteric regulation existing in TrpS from Pyrococcus furiosus (PfTrpS), and how the allosteric conformational ensemble is recovered in laboratory-evolved stand-alone β-subunit variants. We find that recovering the conformational ensemble of a subdomain of TrpS affecting the relative stabilities of open, partially closed, and closed conformations is a prerequisite for enhancing the catalytic efficiency of the β-subunit in the absence of its binding partner. The distal mutations resuscitate the allosterically driven conformational regulation and alter the populations and rates of exchange between these multiple conformational states, which are essential for the multistep reaction pathway of the enzyme. Interestingly, these distal mutations can be a priori predicted by careful analysis of the conformational ensemble of the TrpS enzyme through computational methods. Our study provides the enzyme design field with a rational approach for evolving allosteric enzymes toward improved stand-alone function for biosynthetic applications.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction

“…The results indicate that the relative fraction of the O and PC conformations were virtually identical for both proteins and that the closed conformation was not sampled (Anc2nc-TrpB: 62% O/38% PC/0% C; Anc2nc-TrpB_AM4: 66% O/34% PC/0% C). This is in accordance with the relatively slow O-PC-C transition that usually takes place on time scales larger than the currently accessible simulation times (21).…”

“…Interestingly, the stand-alone variant PfTrpB 0B2 , which has been generated by directed evolution, has virtually identical biochemical properties as Anc2nc-TrpB, including inhibition by PfTrpA (9). In line with these findings, our community analysis of Anc2nc-TrpB and extended MD simulations of PfTrpB 0B2 (21) indicate that allosterically inhibited (or stand-alone) TrpB proteins display an extended conformational heterogeneity of the COMM domain that is normally observed in TS complexes.…”

“…Hence, TrpA binding splits the COMM domain of Anc2nc-TrpB into 2 less-coordinated structural elements, suggesting the perturbance of the productive conformational ensemble required for multistep catalysis. Similarly, enhanced sampling MD techniques have indicated that the inhibitory effect of PfTrpA on the stand-alone PfTrpB 0B2 , which also shows an inversed allosteric effect, can be explained by the truncation of the conformational ensemble and the population of unproductive closed states of the COMM domain (21). In contrast, the COMM domain of the allosterically activated Anc2nc-TrpB_AM4 seems rigidified upon TrpA binding and to recover the conformational heterogeneity required for tryptophan production.…”

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

“…1c and Supplementary Table 2 ). Some of these mutations occurred at residues previously identified as relevant in conformational dynamics (e.g., N167D and S302P) 20 – 22 . Most mutations observed, however, have not been previously identified in laboratory engineering experiments, suggesting that even the consensus of these populations explored uncharacterised regions of Tm TrpB’s fitness landscape, doing so with diversity across replicates (Fig.…”

Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

“…Some of these mutations occurred at residues previously identified as relevant in conformational dynamics (e.g., N167D and S302P). [20][21][22] Most mutations observed, however, have not been previously identified in laboratory engineering experiments, suggesting that even the consensus of these populations explored new regions of TmTrpB's fitness landscape, doing so with diversity across replicates ( Fig. 1c) that might translate to diversity in promiscuous activities across evolved variants.…”