Evolution of Multi-Enzyme Complexes:  The Case of Tryptophan Synthase

Leopoldseder, Sonja; Hettwer, Stefan; Sterner, Reinhard

doi:10.1021/bi061684b

Cited by 21 publications

(43 citation statements)

References 56 publications

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“…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.…”

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confidence: 62%

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

et al. 2014

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ABSTRACT:The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational−structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase β chain/β chain-like TrpB1−TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-L-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the L-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-Lserine.

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confidence: 62%

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

et al. 2014

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“…Moreover, activity titrations have shown that the affinity of sTrpB2i for sTrpA is increased by 2 orders in the presence of L-serine (18). At 60°C, values for K M Ser of 7 mM (Supporting Information Figure S1B) and 40 mM (18) have been determined, independent of the presence of sTrpA. These values are 2 orders of magnitude higher than the intracellular concentration of L-serine found in E. coli (59).…”

Section: Discussionmentioning

confidence: 88%

“…In contrast, the results for the TS from S. solfataricus indicate that the interaction of sTrpA and sTrpB2i is transient and depending on the presence of TrpA and TrpB ligands and that the affinity between the subunits is relatively weak (Table 1). In activity titrations, where catalysis by sTrpA was measured as a function of added sTrpB2i, apparent dissociation constants of 280 nM (with L-serine) and 22 μM (without L-serine) were determined (18). After having stabilized the complex by the ligands GP and L-serine, we now quantified subunit affinity and stoichiometry in the absence of catalytic activity.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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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.

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“…This enzyme has attracted a great deal of attention and been extensively reviewed as the one of the first enzymes to be discovered that catalyzes two distinct reactions at independent active sites (in the a-and b-domains) that are connected via an internal tunnel [274]. This was the first well-documented example of substrate channeling in amino acid biosynthesis.…”

Section: Tryptophan Biosynthesismentioning

confidence: 99%

Amino Acid Biosynthesis

Parker

Pratt

2010

Amino Acids, Peptides and Proteins in Organic Chemistry

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The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter.There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials. The strategic challenge for the biosynthesis of most straight-chain amino acids centers around two issues: how is the a-amino function introduced into the carbon skeleton and what functional group manipulations are required to generate the diversity of side-chain functionality required for the protein function?The core family of straight-chain amino acids does not provide all the functionality required for proteins. a-Amino acids with branched side-chains are used for two purposes; the primary need is related to protein structural issues. Proteins fold into well-defined three-dimensional shapes by virtue of their amphipathic nature: a significant fraction of the amino acid side-chains are of low polarity and the hydrophobic effect drives the formation of ordered structures in which these side-chains are buried away from water. In contrast to the straight-chain amino

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Evolution of Multi-Enzyme Complexes: The Case of Tryptophan Synthase

Cited by 21 publications

References 56 publications

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

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

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

Amino Acid Biosynthesis

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