DEFINING SUBSTRATE SPECIFICITY IN TRYPTOPHAN SYNTHASE BETA‐SUBUNIT HOMOLOGS

Abstract

Pyrococcus furiosus and many other Archaea, hyperthermophilic bacteria, and some plants, possess two trp synthase β subunit genes which encode two separate yet closely related enzymes; TrpB1 and TrpB2. TrpB1s are considered the canonical β-subunit of the trp synthase α2β2 quaternary complex, whereas TrpB2s are a newly discovered homolog. In P. furiosus, TrpB2 is located outside of a full set of trp operon genes, but several Archaea and hyperthermophilic bacteria possess only TrpB2s and no other trp operon genes. The only TrpB2 that has been characterized is that from T. maritima which was found to catalyze a canonical TrpB1 reaction using L-Ser and indole as substrates. We have found that the P. furiosus, T. maritima, and A. aeolicus TrpB2s, as well as the S. typhimirium TrpB1, can utilize L-Ser and L-Cys as substrates in the enzymatic biosynthesis of L-Trp. The P. furiosus TrpB2 appears to follow a biphasic kinetic pattern with two Km’s and two Vmax’s with both substrates. We have applied LC-MS to define the substrate specificity between L-Ser and L-Cys in a competition experiment using β-[13C]-L-Ser. These experiments have revealed that Ser/Cys substrate specificity in the S. typhimirium α2β2 complex is imparted to the β-subunit by the α-subunit, yet in other organisms, like the protozoan Cryptosporidium parvum, which possesses a TrpB1 but no TrpA, the α-subunit is not necessary to discriminate between L-Ser and L-Cys.