Allostery, engineering and inhibition of tryptophan synthase

D'Amico, Rebecca N.; Boehr, David D.

doi:10.1016/j.sbi.2023.102657

Cited by 3 publications

(3 citation statements)

References 49 publications

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“…The visualization of the electrostatic surface properties indicates that this region is devoid of negative charges. The presence of a transient binding site (I2), extended by the binding sites I1, H1 and I3 to the ECH, HAD and KAT active sites, respectively, is consistent with a substrate-channeling mechanism of reaction intermediates between these active sites, as also described in binding studies of Mtb tryptophan synthase (Bosken et al, 2022;D'Amico & Boehr, 2023) and the TS-DHFR bifunctional enzyme (Anderson, 2017;Metzger et al, 2014). Thus, this MtTFE study provides a basis for further studies to better characterize the substrate-channeling properties of MtTFE.…”

Section: Discussionsupporting

confidence: 81%

“…The mechanism of substrate channeling is dependent on the nature of the substrate. For small molecules it involves substrate tunneling through the matrix of the protein, for example in the well studied bifunctional enzyme tryptophan synthase (Barends et al, 2008;Bosken et al, 2022;D'Amico & Boehr, 2023), and for large polar molecules it involves surface crawling (over bulk solvent-exposed surface area), as observed for the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR; Anderson, 2017). In some cases, partial channeling of substrates has been observed (Baker et al, 2012), in which case not all intermediates reach the subsequent active site by surface crawling, but a certain fraction diffuses via bulk solvent to the subsequent active site.…”

Section: Introductionmentioning

confidence: 99%

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Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them

Dalwani,

Metz,

Huschmann

et al. 2024

Acta Crystallogr D Struct Biol

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The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE–fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

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Section: Discussionsupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them

Dalwani,

Metz,

Huschmann

et al. 2024

Acta Crystallogr D Struct Biol

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“…Extensive research is focused on tryptophan synthase (TRPS), the terminal enzyme in the L-tryptophan synthetic pathway [ 1 , 2 , 3 ]. This pathway is crucial for the survival of various microorganisms, including bacteria, plants, and fungi [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

Fungi Tryptophan Synthases: What Is the Role of the Linker Connecting the α and β Structural Domains in Hemileia vastatrix TRPS? A Molecular Dynamics Investigation

Martins,

Viana,

Maigret

2024

Molecules

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Tryptophan synthase (TRPS) is a complex enzyme responsible for tryptophan biosynthesis. It occurs in bacteria, plants, and fungi as an αββα heterotetramer. Although encoded by independent genes in bacteria and plants, in fungi, TRPS is generated by a single gene that concurrently expresses the α and β entities, which are linked by an elongated peculiar segment. We conducted 1 µs all-atom molecular dynamics simulations on Hemileia vastatrix TRPS to address two questions: (i) the role of the linker segment and (ii) the comparative mode of action. Since there is not an experimental structure, we started our simulations with homology modeling. Based on the results, it seems that TRPS makes use of an already-existing tunnel that can spontaneously move the indole moiety from the α catalytic pocket to the β one. Such behavior was completely disrupted in the simulation without the linker. In light of these results and the αβ dimer’s low stability, the full-working TRPS single genes might be the result of a particular evolution. Considering the significant losses that Hemileia vastatrix causes to coffee plantations, our next course of action will be to use the TRPS to look for substances that can block tryptophan production and therefore control the disease.

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Crystallographic fragment binding studies of theMycobacterium tuberculosistrifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate channeling path between them

Dalwani,

Metz,

Huschmann

et al. 2024

Preprint

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TheMycobacterium tuberculosistrifunctional enzyme (MtTFE) is an α2β2tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium, and long chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modelling studies have identified binding sites for the acyl-CoA substrates at the three active sites as well as the NAD+binding pocket at the HAD active site. These studies have also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, in a crystallographic fragment binding study with MtTFE crystals 226 fragments were screened, resulting in the structures of 17 MtTFE-fragment complexes. Analysis of the 143 fragment binding events shows that the ECH active site is the ′binding hotspot′ for the tested fragments, with 50 binding events. The mode of binding of the fragments bound at the active sites provides additional insight on how the long chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 24 fragment binding events between the active sites identify potential transient binding sites of reaction intermediates relevant for possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of these binding sites and to identify substrates for which channeling is crucial.

show abstract

Allostery, engineering and inhibition of tryptophan synthase

Cited by 3 publications

References 49 publications

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them

Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them

Fungi Tryptophan Synthases: What Is the Role of the Linker Connecting the α and β Structural Domains in Hemileia vastatrix TRPS? A Molecular Dynamics Investigation

Crystallographic fragment binding studies of theMycobacterium tuberculosistrifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate channeling path between them

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