The tryptophan synthase (TS) is a linear  complex that catalyzes the last two steps of tryptophan biosynthesis in primary metabolism. It is a model system for subunit interactions in multi-enzyme complexes with a long-standing history of research. The -subunit TrpA and the -subunit TrpB display a rather low catalytic activity in isolation but strongly stimulate each other in the TS complex in an allosteric manner. In this thesis, allostery in the TS was studied by means of two different approaches.
In the first manuscript, the TrpA subunit from Zea mays, which is dependent on activation by the respective TrpB subunit, was compared to a homologue of TrpA, called BX1. BX1 catalyzes the same reaction in secondary metabolism as does TrpA in primary metabolism but is highly active in the absence of a TrpB-like partner protein. The TrpA - BX1 comparison identified two differing amino acids within a loop region known for its role in allosteric activation of TrpA. The transfer of the corresponding BX1 loop residues into TrpA yielded variants with turnover numbers that were increased by at least one order of magnitude and up to 520-fold. This corresponds to the activation of TrpA by TrpB in the wild-type TS complex. At the same time substrate affinity was reduced drastically, an effect that could be reversed by binding of wild-type TrpB to the TrpA variants. These findings contribute to the understanding of the allosteric activation of the -subunit by the -subunit of TS and 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.
In the second manuscript, the allosteric network of TS was analyzed by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. In previous work, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) were computed by means of ASR. The corresponding primordial proteins were produced in Escherichia coli, purified, and characterized. The results showed that LBCA-TS is reminiscent of modern TS by forming a  complex with indole channeling taking place. However, LBCA-TrpA decreases the activity of LBCA-TrpB by a factor of 5 whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis increases the activity of ncTrpB by a factor of 30. In order to identify those amino acid residues that are responsible for this large difference, all six evolutionary TrpA and TrpB intermediates that stepwise link LBCA TS with N. caesariensis TS were produced and characterized. Remarkably, the switching from TrpB-inhibition to TrpB-activation by TrpA occurred between two successive TS intermediates. The comparison of these intermediates and the mutual exchange of residues by iterative rounds of site-directed mutagenesis allowed for the identification of four (out of 413) residues from TrpB that are necessary and sufficient for its allosteric activation by TrpA. These findings demonstrate that ancestral sequence reconstruction can efficiently identify residues essential for allosteric communication and contribute to our understanding of signal propagation in TS.

Die Tryptophan Synthase ist ein linearer abba Komplex, welcher die letzten beiden Schritte der Tryptophan Biosynthese katalysiert. In dieser Arbeit wird anhand der Modellsystems Tryptophan Synthase Allosterie mittels zwei unterschiedlicher Ansätze untersucht.
Im ersten Teil werden mittels paarweisen Sequenzvergleichs zweier homologer rezenter TrpA Enzyme (horizontaler Ansatz) Mutationen in Loop6 von TrpA identifiziert, welche TrpA unabhängig von der allosterischen Aktivierung durch TrpB machen.
Im zweiten Teil werden mittels ASR rekonstruierte Proteine miteinander verglichen (vertikaler Ansatz) um die molekularen Ursachen der Umkehrung der Allosterie in TrpB zu identifizieren.