Ligand-Induced Formation of a Transient Tryptophan Synthase Complex with αββ Subunit Stoichiometry

Abstract

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.

TrpB2 Enzymes are O-Phospho-l-serine Dependent Tryptophan Synthases

“…1 It has been shown that the ssTrpB2i enzyme from Sulfolobus solfataricus forms with ssTrpA a transient, ligand-dependent tryptophan synthase complex having αββ stoichiometry. 8,9 This finding indicates a functional equivalence of TrpB2i and TrpB1 in vivo. In contrast, all TrpB2o and TrpB2a enzymes characterized to date do not interact with TrpA.…”

“…30 It has been shown that saturating concentrations of the ssTrpA ligand glycerol-3-phosphate (GP) and the ssTrpB2i ligand L-serine induce the formation of a transient αββ complex. 8 We tested the consequences of replacing L-serine by O-phospho-L-serine or glycine for complex formation between ssTrpB2i and ssTrpA. Surface plasmon resonance and isothermal titration calorimetry showed that the affinity between ssTrpB2i and ssTrpA was identical, independent of the used ssTrpB2i ligand (Table S3, Supporting Information).…”

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

“…The polysaccharide used in the current study is different from the previous reports in that it is also slowly utilized to some degree through the E. coli maltose-maltodextrin transport system (own unpublished results). The EnBase fed-batch-like medium has been successfully used for high-yield cytoplasmic expression of several non-disulfide bond containing proteins [22-27] as well as functional protein with multiple disulfide bonds [28,29], while in this study we apply this medium for the first time for periplasmic production of disulfide-containing proteins. We also compared two metabolically different E. coli strains regarding their Fab yield in the different growth media.…”

The tryptophan synthase β‐subunit paralogs TrpB1 and TrpB2 in Thermococcus kodakarensis are both involved in tryptophan biosynthesis and indole salvage

“…Among the TrpB2 proteins, only TrpB2o from T. maritima and TrpB2i from S. solfataricus (ssoTrpB2i) have been examined experimentally. It was found that ssoTrpB2i forms a weak and transient complex with TrpA from S. solfataricus (ssoTrpA) only in the presence of the substrate l ‐serine, and activates ssoTrpA unidirectionally . T. kodakarensis harbors a trpB1 gene within the trp operon and a trpB2 gene (tkTrpB2o) outside of the operon.…”