|Hiyama T, Sato T, Imanaka T, Atomi H.
|The tryptophan synthase β-subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage
|archaea; hyperthermophile; trpB; tryptophan; tryptophan synthase
|The last two steps of l-tryptophan (Trp) biosynthesis are catalyzed by Trp synthase, a heterotetramer composed of TrpA and TrpB. TrpB catalyzes the condensation of indole, synthesized by TrpA, and serine to Trp. In the hyperthermophilic archaeon Thermococcus kodakarensis, trpA and trpB (trpB1) are located adjacently in the trpCDEGFB1A operon. Interestingly, several organisms possess a second trpB gene (trpB2) encoding TrpB2, located outside of the trp operon in T. kodakarensis. Until now, the physiological function of trpB2 has not been examined genetically. In the present study, we report the biochemical and physiological analyses of TrpB2 from T. kodakarensis. Kinetic analysis indicated that TrpB2 catalyzed the TrpB reaction but did not interact with TrpA as in the case of TrpB1. When growth phenotypes were examined for gene disruption strains, the double-deletion mutant (ΔtrpB1ΔtrpB2) displayed Trp auxotrophy, whereas individual single mutants (ΔtrpB1 and ΔtrpB2 strains) did not. It has been proposed previously that, in Thermotoga maritima, TrpB2 provides an alternate route to generate Trp from serine and free indole (indole salvage). To accurately examine the capacity of TrpB1 and TrpB2 in Trp synthesis via indole salvage, we constructed ΔtrpEB1 and ΔtrpEB2 strains using strain KUW1 (ΔpyrFΔtrpE) as a host, eliminating the route for endogenous indole synthesis. Indole complemented the Trp auxotrophies of ΔtrpEB1 (ΔpyrFΔtrpEΔtrpB1) and ΔtrpEB2 (ΔpyrFΔtrpEΔtrpB2) to similar levels. The results indicate that TrpB1 and TrpB2 both contribute to Trp biosynthesis in T. kodakarensis and can utilize free indole, and that indole salvage does not necessarily rely on TrpB2 to a greater extent.