Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis β-oxidation trifunctional enzyme

Dalwani, Subhadra; Lampela, Outi; Leprovost, Pierre; Schmitz, W.; Juffer, André H.; Wierenga, Rik K.; Radha, Venkatesan

doi:10.1016/j.jsb.2021.107776

Cited by 8 publications

(30 citation statements)

References 73 publications

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“…Previous crystallographic binding studies of MtTFE with CoA have identified the ECH, HAD and KAT active sites, as well as three additional CoA binding sites, referred to as the CoA-A(HAD/KAT), CoA-B(ECH2) and CoA-C(ECH/HAD) binding sites, based on their location on the surface of the MtTFE tetramer (Dalwani et al ., 2021) ( Table 3, Fig. 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…The mode of binding of CoA at the CoA-A(HAD/KAT), CoA-B(ECH2) and CoA-C(ECH/HAD) binding sites is provided by the coordinates of PDB ID 7O4R (2.8Å resolution, referred to as the CoA-A structure), 7O4S (2.8Å resolution, referred to as the CoA-B structure) and 7O4T (2.1Å resolution, referred to as the CoA-C structure), respectively (Dalwani et al ., 2021). The mode of binding of CoA in the ECH and KAT active sites is also provided by these structures and the CoA-C structure (PDB ID 7O4T) is used as the reference coordinate set.…”

Section: Methodsmentioning

confidence: 99%

“…The crystal structure of MtTFE in its unliganded and CoA bound forms identified the binding sites for its CoA-conjugated substrates at each of its three active sites (CoA(ECH), CoA(HAD) and CoA(KAT)) (Venkatesan & Wierenga, 2013) as well as the binding site for the NAD + co-factor at the HAD active site (Dalwani et al ., 2021). A structural analysis of MtTFE highlighted the presence of positively charged residues between the ECH and HAD active sites and a neutral surface path between the HAD and KAT active sites (Dalwani et al ., 2021; Venkatesan & Wierenga, 2013). These crystallographic binding studies revealed also three additional CoA binding sites on the surface of MtTFE, referred to as the CoA-B(ECH2), CoA-A(HAD/KAT) and CoA-C(ECH/HAD) sites (Dalwani et al ., 2021) ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…A structural analysis of MtTFE highlighted the presence of positively charged residues between the ECH and HAD active sites and a neutral surface path between the HAD and KAT active sites (Dalwani et al ., 2021; Venkatesan & Wierenga, 2013). These crystallographic binding studies revealed also three additional CoA binding sites on the surface of MtTFE, referred to as the CoA-B(ECH2), CoA-A(HAD/KAT) and CoA-C(ECH/HAD) sites (Dalwani et al ., 2021) ( Fig. 1 ).…”

Section: Introductionmentioning

confidence: 99%

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Self Cite

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show abstract

“…These proteins exhibit high amino acid identities in all regions related to catalysis as outlined above, but especially around positions 119–129 and 194–208, which are involved in tightly binding the first CoA cofactor. Although the location of the former motif is equivalent to that of general CoA‐binding loops in other thiolases, the extensive conformation changes and final shapes of the two motifs in free and CoA‐bound BbsB are unique and have not been observed in any other thiolases [12,34]. The classical homodi‐ or ‐tetrameric thiolases normally use helical segments of varying orientation and totally different amino acid sequences as covering loops to bind CoA‐thioesters into a pre‐formed cavity [12,18], instead of the two highly mobile loop segments linked with BbsA.…”

Section: Discussionmentioning

confidence: 99%

Finis tolueni: a new type of thiolase with an integrated Zn‐finger subunit catalyzes the final step of anaerobic toluene metabolism

et al. 2022

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Anaerobic toluene degradation involves β‐oxidation of the first intermediate (R)‐2‐benzylsuccinate to succinyl‐CoA and benzoyl‐CoA. Here, we characterize the last enzyme of this pathway, (S)‐2‐benzoylsuccinyl‐CoA thiolase (BbsAB). Although benzoylsuccinyl‐CoA is not available for enzyme assays, the recombinantly produced enzymes from two different species showed the reverse activity, benzoylsuccinyl‐CoA formation from benzoyl‐CoA and succinyl‐CoA. Activity depended on the presence of both subunits, the thiolase family member BbsB and the Zn‐finger protein BbsA, which is affiliated to the DUF35 family of unknown function. We determined the structure of BbsAB from Geobacter metallireducens with and without bound CoA at 1.7 and 2.0 Å resolution, respectively. CoA binding into the well‐known thiolase cavity triggers an induced‐fit movement of the highly disordered covering loop, resulting in its rigidification by forming multiple interactions to the outstretched CoA moiety. This event is coupled with an 8 Å movement of an adjacent hairpin loop of BbsB and the C‐terminal domain of BbsA. Thereby, CoA is placed into a catalytically productive conformation, and a putative second CoA binding site involving BbsA and the partner BbsB′ subunit is simultaneously formed that also reaches the active center. Therefore, while maintaining the standard thioester‐dependent Claisen‐type mechanism, BbsAB represents a new type of thiolase.

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Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex

Son¹,

Ahn²,

Hong³

et al. 2022

Archives of Biochemistry and Biophysics

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Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis β-oxidation trifunctional enzyme

Cited by 8 publications

References 73 publications

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

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

Finis tolueni: a new type of thiolase with an integrated Zn‐finger subunit catalyzes the final step of anaerobic toluene metabolism

Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex

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