Alternating Sites Reactivity Is a Common Feature of Thiamin Diphosphate-Dependent Enzymes As Evidenced by Isothermal Titration Calorimetry Studies of Substrate Binding

Schröder-Tittmann, Kathrin; Meyer, Danilo; Arens, Johannes; Wechsler, Cindy; Tietzel, Michael; Golbik, Ralph; Tittmann, Kai

doi:10.1021/bi301591e

Cited by 16 publications

(14 citation statements)

References 26 publications

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“…In the process we also provide evidence of the half-of-sites, or alternating sites, reactivity that is postulated to be a common feature of all ThDP-dependent enzymes (Schroder-Tittmann et al, 2013).…”

Section: Introductionmentioning

confidence: 67%

“…Evidence for active-site coupling, giving rise to alternating site reactivity in ThDP-dependent enzymes, was first found in benzoylformate decarboxylase (Sergienko et al, 2000), and is now believed to occur in many such enzymes (Frank et al, 2007;Schroder-Tittmann et al, 2013). Nevertheless, crystal structures of ThDP-dependent enzymes often fail to show evidence of active-site asymmetry, perhaps due to crystal packing constraints or ligand saturation during crystallization.…”

Section: Active-site Couplingmentioning

confidence: 99%

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Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

et al. 2016

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Menaquinone (MQ) is an essential component of the respiratory chains of many pathogenic organisms, including Mycobacterium tuberculosis (Mtb). The first committed step in MQ biosynthesis is catalyzed by 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD), a thiamin diphosphate (ThDP)-dependent enzyme. Catalysis proceeds through two covalent intermediates as the substrates 2-oxoglutarate and isochorismate are successively added to the cofactor before final cleavage of the product. We have determined a series of crystal structures of Mtb-MenD that map the binding of both substrates, visualizing each step in the MenD catalytic cycle, including both intermediates. ThDP binding induces a marked asymmetry between the coupled active sites of each dimer, and possible mechanisms of communication can be identified. The crystal structures also reveal conformational features of the two intermediates that facilitate reaction but prevent premature product release. These data fully map chemical space to inform early-stage drug discovery targeting MenD.

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Section: Introductionmentioning

confidence: 67%

Section: Active-site Couplingmentioning

confidence: 99%

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

et al. 2016

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“…The two TPP cofactors in the functional (ab) 2 heterotetramer are located at relatively close positions (16.5 Å for the nearest atoms), suggesting a possible structural influence between the two active sites. Kinetic and structural studies on various thiamine-dependent enzymes suggested a communication (alternating sites reactivity) between the cofactors in different protomers 31 32 33 .…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of archaeal 2-oxoacid:ferredoxin oxidoreductases from Sulfolobus tokodaii

Yan

Maruyama

Arakawa

et al. 2016

Sci Rep

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As the first three-dimensional structure of the two-subunit type 2-oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn2+-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins.

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“…thought that there is considerable communication between active sites in order to coordinate the ping-pong kinetics between active sites, such that catalysis alternates between active sites, giving rise to the 'half-of-the-sites reactivity' phenomenon that has been observed in transketolase 7 and other TPP-dependent enzymes [8][9][10][11] .…”

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confidence: 99%

The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

Wilkinson

Dalby

2020

Sci Rep

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We recently characterised a low-activity form of E. coli transketolase, tK low , which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TK high , in the presence of Mg 2+. We observed previously that partial oxidation was responsible for increased tK high activity, while lowactivity tK low was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the β-hydroxypyruvate (HpA) donor substrate to wild-type transketolase and a variant, S385Y/D469T/R520Q, that is active towards aromatic aldehydes. Transketolase HPA affinity again revealed the two distinct forms of transketolase at a TK high :tK low ratio that matched those observed previously via tpp binding to each variant. the HpA dissociation constant of tK low was comparable to the substrate-inhibition dissociation constant, K i HPA , determined previously. We provide evidence that K i HPA is a convolution of binding to the low-activity tK low-tK low dimer, and the tK low subunit of the partially-active tK high-tK low mixed dimer, where HpA binding to the tK low subunit of the mixed dimer results in inhibition of the active tK high subunit. Heat-activation of transketolase was similarly investigated and found to convert the tK low subunit of the mixed dimer to have tK high-like properties, but without oxidation. Transketolase is a key enzyme of the pentose phosphate pathway (PPP), is ubiquitous in all organisms, and provides a unique link between glycolysis and the non-oxidative phase of the PPP. Transketolase is a thiamine pyrophosphate (TPP)-dependent enzyme that reversibly transfers a two-carbon ketol group from a donor substrate (usually a five-carbon ketose) to an acceptor aldehyde substrate (usually a five-carbon aldose) via a ping-pong reaction mechanism, forming a new asymmetric CC bond with high regio-and stereo-specificity. In biocatalysis, β-hydroxypyruvate (HPA) is often used as the donor substrate due to the irreversible, concomitant release of CO 2 as a by-product. Strong substrate inhibition has been observed above 25 mM HPA with an inhibition constant of around 42 mM 1,2. The cause of this substrate inhibition is currently unknown and is addressed in this study. The inactive, apo-form of transketolase is in a monomer-dimer equilibrium that is dependent on protein concentration. Upon cofactor binding, both the inactive apo-monomer and apo-dimer are converted into the catalytically active, dimeric holo-form of, until recently, seemingly structurally-identical subunits, with two active-sites per homodimer, located at the subunit interface 3,4. Each active site is comprised of one divalent cation, such as Mg 2+ , and one TPP molecule. In the S. cerevisiae transketolase apo-dimer, even after removal of free Ca 2+ , one Ca 2+ ion was bound extremely tightly to one active site and could only be removed using harsh treatment, while the second Ca 2+ ion dissociated easily. Subs...

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Alternating Sites Reactivity Is a Common Feature of Thiamin Diphosphate-Dependent Enzymes As Evidenced by Isothermal Titration Calorimetry Studies of Substrate Binding

Cited by 16 publications

References 26 publications

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

Structural Views along the Mycobacterium tuberculosis MenD Reaction Pathway Illuminate Key Aspects of Thiamin Diphosphate-Dependent Enzyme Mechanisms

Crystal structures of archaeal 2-oxoacid:ferredoxin oxidoreductases from Sulfolobus tokodaii

The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

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