Entropic stabilization of the tryptophan synthase α-subunit from a hyperthermophile, Pyrococcus furiosus : X-ray analysis and calorimetry

This research was originally published in Journal of Biological Chemistry. Yuriko Yamagata, Kyoko Ogasahara, Yusaku Hioki, Soo Jae Lee, Atsushi Nakagawa, Haruki Nakamura, Masami Ishida, Seiki Kuramitsui, and Katsuhide Yutani. Entropic stabilization of the tryptophan synthase α-subunit from a hyperthermophile, Pyrococcus furiosus : X-ray analysis and calorimetry. J. Biol. Chem. 2001; 276, 11062-11071. © the American Society for Biochemistry and Molecular Biology

The Role of Oligomerization and Cooperative Regulation in Protein Function: The Case of Tryptophan Synthase

The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of α/β–dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand–bound conformations. Our simulations also revealed that the α/β–dimeric unit stabilizes the substrate–protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope

Structural insights into the basis and evolution of interactions in multi-subunit protein assemblies. tryptophan synthase and titin FNIII-repeats

Cellular processes benefit from evolutionary shaping when optimized protein-protein interactions result in enhanced functionality. In fact, most cellular proteins are tightly embedded into biological networks that function following a modularity principle. Modularity, whether based on components as parts of stable protein complexes or as dynamic units that interact only transiently (as in signalling and metabolic cascades), facilitates the combinatorial generation of complexity in protein networks through the re-wiring of modules in addition to the diversification of individual proteins – thereby increasing the “evolvability” of the system. The mechanisms that drive the emergence and evolution of molecular recognition in protein networks remain unclear. It is difficult to justify such evolution on the basis of organismic advantage, since the latter might only be noticeable once full pathways and cascades have evolved. It is then likely that the evolution of protein-protein interactions is in the first instance driven by a molecular principle of local advantage to the protein system itself - for example, molecular stability. Unfortunately, it is difficult to gain insights into the evolution of protein-protein interactions since the pathways of evolutionary shaping normally let intermediates of evolution disappear. Subsequently, conclusions are more usually drawn from the comparison of proteins between different species and by mutagenesis probing. In the current study, we aim at gaining an insight into the evolutionary shaping of proteins surfaces for hetero-complex formation by studying two systems at an early stage of development: Tryptophan Synthase B2b (TrpB2b) from S. solfataricus and the modular interfaces of the poly-FNIII tandems in the muscle filament titin. In the case of TrpB2b, the evolution of inter-subunit communication is addressed in addition. Both structures have been elucidated using X-ray crystallography and a comparative analysis of their surfaces has been carried out. The architectural elements subjected to evolutionary pressure have been identified and conclusions on their relation to function and evolution have been drawn

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

BACKGROUND: To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1). RESULTS: aaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80°C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + P(i )with k(cat )= 5 × 10(-3 )s(-1 )and K(m )= 5.5 × 10(-6 )M. In addition, the enzyme exhibits GTPase activity (k(cat )= 9 × 10(-3 )s(-1 )and K(m)= 45 × 10(-6 )M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally. CONCLUSION: Since aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases

Modelling the evolution of the archeal tryptophan synthase

BACKGROUND: Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate. RESULTS: In order to study the evolution of trp genes, completely sequenced archeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o) contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operon-based trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i) consisted of trpB2 genes located inside, the other (trpB2_a) of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium. CONCLUSION: In archeal genomes, several stages of trpB evolution, TrpA/TrpB cooperation, and operon formation can be observed. Thus, archeal trp genes may serve as a model system for studying the evolution of protein-protein interactions and operon formation

A biophysical study of the thermostability of citrate synthase

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN059683 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Partially redundant tryptophan synthase and MYB transcription factor genes regulate indolic defense compound synthesis in Arabidopsis thaliana

In the model cruciferous plant, Arabidopsis thaliana, tryptophan (Trp) is a focal point for growth and defense as it is used for the production of secondary metabolites including the growth hormone indole-3-acetic acid (IAA, or auxin), and two classes of defense compounds: indole glucosinolates (IGs) and camalexin. Trp metabolism in plants is of general importance to agriculture because animals (including humans) cannot synthesize Trp and must obtain it from their diet. Questions remain about the synthesis and regulation of Trp and how it relates to secondary metabolism in Arabidopsis. In this thesis it is shown that IGs are a sink for Trp metabolism because auxotrophic mutants deficient in Trp production are suppressed in combination with the IG-deficient cyp79B2 cyp79B3 mutant and enhanced in combination with IG overproducing mutant, atr1D. Because Trp auxotrophic mutants were found to produce IGs, the four predicted Arabidopsis Trp Synthase Beta genes (TSB1, TSB2, TSB3 and TSBt2) were examined for their role in Trp primary and secondary metabolism. It was determined that members of this gene family, while being redundant for enzyme activity, may have unique functions in channeling Trp to different secondary endpoints. tsb1 tsb2 plants display a healthier phenotype and produce lower IG levels than the single tsb1 mutants, in contrast to tsb1 tsbt2 plants, which have elevated IG production and an enhanced auxotrophic phenotype. tsb2 tsbt2 plants are indiscernible from WT. Gene expression in Trp biosynthetic pathway steps, IG biosynthesis genes, and regulatory TFs is dysregulated in these mutants. In a second part of this thesis, transcriptional regulation of IG synthesis was examined with respect to tissue specificity and stress. In collaboration with Judith Bender's laboratory at Brown University, the function of a subfamily of three Myb transcription factors that have been implicated in regulating IG biosynthesis genes was studied. Using combinations of Myb knockout mutants and GUS reporter plants, tissue specific roles for MYB34 and MYB51 in root and shoot tissues, respectively, were found. In addition, roles were discovered for MYB34 in mediating anti-herbivory signals, and for both MYB51 and MYB122 in regulating defense against microbial pathogens

A comprehensive analysis of the importance of translation initiation factors for Haloferax volcanii applying deletion and conditional depletion mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed