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

Abstract

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA fromNeptuniibacter caesariensisallosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Transcriptional factor engineering in microbes for industrial biotechnology

“…Based on the analysis of the currently existing phylogenic descendants ADH1 (ethanol generation) and ADH2 (ethanol consumption), an inferred and constructed ancestral ADH had the functions of both descendants 45 . ASR can be used to design mutants with a higher degree of promiscuity and less specificity than the currently existing enzymes, 46 to engineer the thermostability of proteins, 47,48 and to elucidate the molecular mechanism of enzymes 49,50 …”

“…45 ASR can be used to design mutants with a higher degree of promiscuity and less specificity than the currently existing enzymes, 46 to engineer the thermostability of proteins, 47,48 and to elucidate the molecular mechanism of enzymes. 49,50 A limited number of mutations at the amino acid level can cause significant differences in protein properties, which is also the case of naturally existing ancestral transcription factor PmrA. 51 These facts enable us to envision that the TFs designed by following the ancestral sequence reconstruction method will dramatically alter the regulatory network in a cell.…”

Rational Prediction of Distal Activity-Enhancing Mutations in Tryptophan Synthase

“…[39][40][41] In a previous work, we reconstructed the TrpS phylogenetic tree and identified a shift in the allosteric modulation exerted by TrpA on TrpB activity. 42,43 The analysis of the steady state kinetic parameters of the last bacterial common ancestor (LBCA) revealed high stand-alone activity of LBCA-TrpB and its allosteric inhibition in the presence of TrpA. Along the phylogenetic tree, this inhibition was gradually inversed towards allosteric activation existing in modern TrpB (Figure 1c).…”

“…Our study presents a computational enzyme design approach that is not restricted to active site mutations and demonstrates that the challenge to rationally predict distal mutations can be ultimately addressed by exploring the conformational energy landscape of enzymes in combination with cross correlation and bioinformatic tools. 42 Numbers next to each edge indicate the number of mutations accumulated in TrpA and TrpB with respect to the previous node. While LBCA-TrpB gets deactivated by TrpA and exhibits stand-alone function, the allosteric effect of TrpA is reverted along the phylogenetic tree with a switch between ANC2 TrpB and ANC3 TrpB to an allosteric activation, as observed in extant ncTrpB.…”

Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome

“…These findings encouraged us to explore ancestral sequence reconstruction (ASR) ( Gumulya and Gillam, 2017 ; Merkl and Sterner, 2016 ; Risso and Sanchez-Ruiz, 2017 ; Thornton, 2004 ; Wilding et al., 2017 ) to engineer lysosomal sulfatases. ASR is a bioinformatics method that has yielded valuable insight into enzyme evolution ( Nguyen et al., 2016 ; Risso et al., 2013 ) and protein structure-function relationships ( Nicoll et al., 2020 ; Schupfner et al., 2020 ; Wilson et al., 2015 ), but has rarely been applied in the context of biopharmaceuticals. One exception is the engineering of coagulation factor VIII with improved expression rates and decreased decay rates, allowing for lower doses in gene therapy experiments toward treatment of hemophilia A ( Samelson-Jones and Arruda, 2019 ; Zakas et al., 2017 ).…”