BX1 from Zea mays (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase α-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS. Using cross-linking and mutagenesis, we identified the physiological dimerization interface of zmBX1. We found that homodimerization has only minor effects on catalysis and stability. A comparison of the zmBX1-zmBX1 homodimer and zmTrpA-zmTrpB heterodimer interfaces suggest that homodimerization in zmBX1 might, at an early point in evolution, have served as a mechanism to exclude the interaction with the tryptophan synthase β-subunit (zmTrpB), marking its transition from primary to secondary metabolism.

The manuscript describes the use of chemical cross-linking/mass spectrometry and mutagenesis to investigate the dimeric interface of the tryptophan synthase α-subunit homolog, BX1. This work indicates that BX1 homodimerization might have served as a mechanism to exclude an interaction with the tryptophan synthase β-subunit, TrpB, at an early time in evolution, thereby eliminating cross-talk between primary and secondary metabolism. This work would be of interest to mass spectrometrists and structural biologist as it presents a workflow to determine the physiological protein-protein interactions within crystal structures using chemical cross-linking/mass spectrometry and mutagenesis as complementary structural biology techniques, thereby eliminating ambiguity and potential mis-assignments due to the presence of additional (artificial) protein contacts formed during the crystallization process.

The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α‐subunit TrpA is stimulated several hundredfold through the interaction with the β‐subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active‐site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated “stand‐alone” TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self‐reliant, stand‐alone, secondary metabolic enzyme.

Significance Enzyme complexes consist of several protein subunits that often catalyze sequential reactions in the cell. The activities of the individual subunits must be kept in phase, which requires a sophisticated communication mechanism that is mediated by certain residues. We wanted to elucidate the communication mechanisms between the α-subunits and β-subunits of the tryptophan synthase (TS) complex, which catalyzes the last 2 steps of the biosynthesis of the essential amino acid tryptophan. To this end, an approach involving the reconstruction of TSs from extinct species was followed. The results of our experimental and computational analyses identified 4 residues that contribute to the communication between the α-subunits and β-subunits and provide an explanation of how they act.

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions-deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape "minima" in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.