Allosteric regulation of catalytic activity is a widespread property of multi-enzyme complexes. The tryptophan synthase is a prototypical allosteric enzyme where the constituting α (TrpA) and β (TrpB) subunits mutually activate each other in a manner that is incompletely understood. Experimental and computational studies have shown that LBCA-TrpB from the last bacterial common ancestor contains six residues (Res6) distal from the active site that allow for high stand-alone catalytic activity in the absence of a TrpA subunit. In the present study, a database search revealed that Res6 is also present in the extant plTrpB from Pelodictyon luteolum. The plTrpB enzyme showed a high stand-alone activity and only a moderate activation by plTrpA. The replacement of LBCA-Res6 in plTrpB with the consensus residues from a multiple sequence alignment yielded plTrpB-con, which showed a dramatically decreased stand-alone activity but was strongly stimulated by plTrpA. These findings suggest that the effect of these six key allosteric residues is largely independent of the protein context within a specific TrpB enzyme. Analysis of the conformational landscapes of plTrpB and plTrpB-con revealed that plTrpB in isolation displays efficient closure of both the active site and the communication (COMM) domain. In contrast, these catalytically competent states are destabilized in plTrpB-con but can be recovered by the addition of plTrpA. A correlation-based shortest path map (SPM) analysis reveals that the catalytically and allosterically relevant domains—specifically, the COMM domain in TrpB and loops 2 and 6 in TrpA—are tightly interconnected exclusively in plTrpA:plTrpB-con.