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Carlo Ignazio Giovanni Tuberoso - One of the best experts on this subject based on the ideXlab platform.
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traceability of satsuma mandarin citrus unshiu marc honey through nectar honey sac honey pathways of the headspace volatiles and semi volatiles chemical markers
Molecules, 2016Co-Authors: Igor Jerković, Zvonimir Marijanovic, Marina Zekic, Lidija Svečnjak, Saša Prđun, Dragan Bubalo, Carlo Ignazio Giovanni TuberosoAbstract:Headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by GC-MS/FID, were applied for monitoring the nectar (NE)/honey-sac (HoS)/honey (HO) pathways of the headspace, volatiles, and semi-volatiles. The major NE (4 varieties of Citrus unshiu) headspace compounds were linalool, α-terpineol, 1H-indole, methyl Anthranilate, and phenylacetonitrile. Corresponding extracts contained, among others, 1H-indole, methyl Anthranilate, 1,3-dihydro-2H-indol-2-one and caffeine. The major HoS headspace compounds were linalool, α-terpineol, 1,8-cineole, 1H-indole, methyl Anthranilate, and cis-jasmone. Characteristic compounds from HoS extract were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl Anthranilate, and phenylacetonitrile. However, HO headspace composition was significantly different in comparison to NE and HoS with respect to phenylacetaldehyde and linalool derivatives abundance that appeared as the consequence of the hive conditions and the bee enzyme activity. C. unshiu honey traceability is determined by chemical markers: phenylacetaldehyde, phenylacetonitrile, linalool and its derivatives, as well as 1H-indole, 1,3-dihydro-2H-indol-2-one, and caffeine.
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Traceability of Satsuma Mandarin (Citrus unshiu Marc.) Honey through Nectar/Honey-Sac/Honey Pathways of the Headspace, Volatiles, and Semi-Volatiles: Chemical Markers
Molecules (Basel Switzerland), 2016Co-Authors: Igor Jerković, Lidija Svečnjak, Saša Prđun, Dragan Bubalo, Zvonimir Marijanović, Marina Zekić, Carlo Ignazio Giovanni TuberosoAbstract:Headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by GC-MS/FID, were applied for monitoring the nectar (NE)/honey-sac (HoS)/honey (HO) pathways of the headspace, volatiles, and semi-volatiles. The major NE (4 varieties of Citrus unshiu) headspace compounds were linalool, α-terpineol, 1H-indole, methyl Anthranilate, and phenylacetonitrile. Corresponding extracts contained, among others, 1H-indole, methyl Anthranilate, 1,3-dihydro-2H-indol-2-one and caffeine. The major HoS headspace compounds were linalool, α-terpineol, 1,8-cineole, 1H-indole, methyl Anthranilate, and cis-jasmone. Characteristic compounds from HoS extract were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl Anthranilate, and phenylacetonitrile. However, HO headspace composition was significantly different in comparison to NE and HoS with respect to phenylacetaldehyde and linalool derivatives abundance that appeared as the consequence of the hive conditions and the bee enzyme activity. C. unshiu honey traceability is determined by chemical markers: phenylacetaldehyde, phenylacetonitrile, linalool and its derivatives, as well as 1H-indole, 1,3-dihydro-2H-indol-2-one, and caffeine.
Everett C Pesci - One of the best experts on this subject based on the ideXlab platform.
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structure of pqsd a pseudomonas quinolone signal biosynthetic enzyme in complex with Anthranilate
Biochemistry, 2009Co-Authors: Asim K Bera, J P Coleman, Everett C Pesci, Vesna Atanasova, Howard Robinson, Edward Eisenstein, James F ParsonsAbstract:Pseudomonas quinolone signal (PQS), 2-heptyl-3-hydroxy-4-quinolone, is an intercellular alkyl quinolone signaling molecule produced by the opportunistic pathogen Pseudomonas aeruginosa. Alkyl quinolone signaling is an atypical system that, in P. aeruginosa, controls the expression of numerous virulence factors. PQS is synthesized from the tryptophan pathway intermediate, Anthranilate, which is either derived from the kynurenine pathway or from an alkyl quinolone specific Anthranilate synthase encoded by phnAB. Anthranilate is converted to PQS by the enzymes encoded by the pqsABCDE operon and pqsH. PqsA forms an activated anthraniloyl-CoA thioester that shuttles Anthranilate to the PqsD active site where it is transferred to Cys112 of PqsD. In the only biochemically characterized reaction, a condensation then occurs between anthraniloyl-PqsD and malonyl-CoA or malonyl-ACP, a second PqsD substrate, forming 2,4-dihydroxyquinoline (DHQ). The role PqsD plays in the biosynthesis of other alkyl quinolones, such as PQS, is unclear though it has been reported to be required for their production. No evidence however, exists that DHQ is a PQS precursor. Here we present a structural and biophysical characterization of PqsD that includes several crystal structures of the enzyme including that of the PqsD-Anthranilate covalent intermediate and the inactive Cys112Ala active site mutant in complex with Anthranilate. The structure reveals that PqsD is structurally similar to the FabH and chalcone synthase families of fatty acid and polyketide synthases. The crystallographic asymmetric unit contains a PqsD dimer. The PqsD monomer is composed of two nearly identical ~170 residue αβαβα domains. The structures show Anthranilate-liganded Cys112 is positioned deep in the protein interior at the bottom of a ~15 A long channel while a second anthraniloyl-CoA molecule is waiting in the cleft leading to the protein surface. Cys112, His257, and Asn287 form the FabH-like catalytic triad of PqsD. The C112A mutant is inactive although it still reversibly binds anthraniloyl-CoA. The covalent complex between Anthranilate and Cys112 clearly illuminates the orientation of key elements of the PqsD catalytic machinery and represents a snapshot of a key point in the catalytic cycle.
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pseudomonas aeruginosa pqsa is an Anthranilate coenzyme a ligase
Journal of Bacteriology, 2008Co-Authors: J P Coleman, Worth M Calfee, John M Farrow, Lynn L Hudson, Susan L Mcknight, Claire A Lindsey, Everett C PesciAbstract:Pseudomonas aeruginosa is an opportunistic human pathogen which relies on several intercellular signaling systems for optimum population density-dependent regulation of virulence genes. The Pseudomonas quinolone signal (PQS) is a 3-hydroxy-4-quinolone with a 2-alkyl substitution which is synthesized by the condensation of anthranilic acid with a 3-keto-fatty acid. The pqsABCDE operon has been identified as being necessary for PQS production, and the pqsA gene encodes a predicted protein with homology to acyl coenzyme A (acyl-CoA) ligases. In order to elucidate the first step of the 4-quinolone synthesis pathway in P. aeruginosa, we have characterized the function of the pqsA gene product. Extracts prepared from Escherichia coli expressing PqsA were shown to catalyze the formation of anthraniloyl-CoA from Anthranilate, ATP, and CoA. The PqsA protein was purified as a recombinant His-tagged polypeptide, and this protein was shown to have Anthranilate-CoA ligase activity. The enzyme was active on a variety of aromatic substrates, including benzoate and chloro and fluoro derivatives of Anthranilate. Inhibition of PQS formation in vivo was observed for the chloro- and fluoroAnthranilate derivatives, as well as for several analogs which were not PqsA enzymatic substrates. These results indicate that the PqsA protein is responsible for priming Anthranilate for entry into the PQS biosynthetic pathway and that this enzyme may serve as a useful in vitro indicator for potential agents to disrupt quinolone signaling in P. aeruginosa.
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two distinct pathways supply Anthranilate as a precursor of the pseudomonas quinolone signal
Journal of Bacteriology, 2007Co-Authors: John M Farrow, Everett C PesciAbstract:Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised patients and those with cystic fibrosis (CF). This gram-negative bacterium uses multiple cell-to-cell signals to control numerous cellular functions and virulence. One of these signals is 2-heptyl-3-hydroxy-4-quinolone, which is referred to as the Pseudomonas quinolone signal (PQS). This signal functions as a coinducer for a transcriptional regulator (PqsR) to positively control multiple virulence genes and its own synthesis. PQS production is required for virulence in multiple models of infection, and it has been shown to be produced in the lungs of CF patients infected by P. aeruginosa. One of the precursor compounds from which PQS is synthesized is the metabolite Anthranilate. This compound can be derived from the conversion of chorismate to Anthranilate by an Anthranilate synthase or through the degradation of tryptophan via the Anthranilate branch of the kynurenine pathway. In this study, we present data which help to define the kynurenine pathway in P. aeruginosa and show that the kynurenine pathway serves as a critical source of Anthranilate for PQS synthesis. We also show that the kyn pathway genes are induced during growth with tryptophan and that they are autoregulated by kynurenine. This study provides solid foundations for the understanding of how P. aeruginosa produces the Anthranilate that serves as a precursor to PQS and other 4-quinolones.
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interference with pseudomonas quinolone signal synthesis inhibits virulence factor expression by pseudomonas aeruginosa
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Worth M Calfee, J P Coleman, Everett C PesciAbstract:Pseudomonas aeruginosa is an opportunistic pathogen that controls numerous virulence factors through intercellular signals. This bacterium has two quorum-sensing systems (las and rhl), which act through the intercellular signals N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL), respectively. P. aeruginosa also produces a third intercellular signal that is involved in virulence factor regulation. This signal, 2-heptyl-3-hydroxy-4-quinolone [referred to as the Pseudomonas quinolone signal (PQS)], is a secondary metabolite that is part of the P. aeruginosa quorum-sensing hierarchy. PQS can induce both lasB (encodes LasB elastase) and rhlI (encodes the C4-HSL synthase) in P. aeruginosa and is produced maximally during the late stationary phase of growth. Because PQS is an intercellular signal that is part of the quorum-sensing hierarchy and controls multiple virulence factors, we began basic studies designed to elucidate its biosynthetic pathway. First, we present data that strongly suggest that Anthranilate is a precursor for PQS. P. aeruginosa converted radiolabeled Anthranilate into radioactive PQS, which was bioactive. We also found that an Anthranilate analog (methyl Anthranilate) would inhibit the production of PQS. This analog was then shown to have a major negative effect on elastase production by P. aeruginosa. These data provide evidence that precursors of intercellular signals may provide viable targets for the development of therapeutic treatments that will reduce P. aeruginosa virulence.
Emily J. Parker - One of the best experts on this subject based on the ideXlab platform.
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The Substrate Capture Mechanism of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Provides a Mode for Inhibition
2016Co-Authors: Alina Castell, Esther M M Bulloch, Edward N. Baker, Emily J. Parker, Francesca L Short, Dmitri D A Joseph, Clare E Lee, Genevieve L. Evans, Tammie V. M. Cookson, Shaun J. LottAbstract:Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5′-phosphoribosyl 1′-pyrophosphate (PRPP) and Anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two Anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic Anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5–119 μM, with the strongest inhibition displayed by a biAnthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme’s activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action
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structures of mycobacterium tuberculosis Anthranilate phosphoribosyltransferase variants reveal the conformational changes that facilitate delivery of the substrate to the active site
Biochemistry, 2015Co-Authors: T V M Cookson, Genevieve L. Evans, Alina Castell, Edward N. Baker, J S Lott, Emily J. ParkerAbstract:Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and Anthranilate. The roles of residues predicted to be involved in Anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both Anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of Anthranilate to bind in three sites along an extended Anthranilate tunnel and expose the role of the mobile β2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The β2-α6 loop moves sequentially between a "folded" conformation, partially occluding the Anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows Anthranilate access via the tunnel to the active site. The return of the β2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind Anthranilate at the opening of the Anthranilate tunnel for subsequent reactions. Multiple Anthranilate molecules blocking the Anthranilate tunnel prevent the β2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme.
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Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site
2015Co-Authors: Tammie V. M. Cookson, Shaun J. Lott, Genevieve L. Evans, Alina Castell, Edward N. Baker, Emily J. ParkerAbstract:Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg2+-dependent reaction between 5′-phosphoribosyl-1′-pyrophosphate (PRPP) and Anthranilate. The roles of residues predicted to be involved in Anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both Anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of Anthranilate to bind in three sites along an extended Anthranilate tunnel and expose the role of the mobile β2−α6 loop in facilitating the enzyme’s sequential reaction mechanism. The β2−α6 loop moves sequentially between a “folded” conformation, partially occluding the Anthranilate tunnel, via an “open” position to a “closed” conformation, which supports PRPP binding and allows Anthranilate access via the tunnel to the active site. The return of the β2−α6 loop to the “folded” conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind Anthranilate at the opening of the Anthranilate tunnel for subsequent reactions. Multiple Anthranilate molecules blocking the Anthranilate tunnel prevent the β2−α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme
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alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by Anthranilate phosphoribosyltransferase from mycobacterium tuberculosis
Biochemical Journal, 2014Co-Authors: T V M Cookson, Esther M M Bulloch, Shaun J. Lott, Genevieve L. Evans, Alina Castell, Edward N. Baker, Francesca L Short, Emily J. ParkerAbstract:AnPRT (Anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis ( Mtb ). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5′-phosphoribosyl-1′-pyrophosphate) to Anthranilate to form PRA (5′-phosphoribosyl Anthranilate). Mtb -AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by Anthranilate was observed. Antimycobacterial fluoroAnthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb -AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with Anthranilate analogues reveal two distinct binding sites for Anthranilate. One site is located over 8 A (1 A=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site Anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for Anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner Anthranilate-binding site. It is proposed that Anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP. Abbreviations: AnPRT, Anthranilate phosphoribosyltransferase; Eco, Escherichia coli; FA, fluoroAnthranilate; F-PRA, fluoro-PRA; InGP, indole glycerol phosphate; InGPS, indole glycerol phosphate synthase; KIE, kinetic isotope effect; MA, methylAnthranilate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; M-PRA, methyl-PRA; Mtb, Mycobacterium tuberculosis; NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PR, 5′-phosphoribosyl; PRA, 5′-phosphoribosyl Anthranilate; PRAI, 5′-phosphoribosyl Anthranilate isomerase; PRPP, 5′-phosphoribosyl-1′-pyrophosphate; PRT, phosphoribosyltransferase; Sso, Sulfolobus solfataricus; TB, tuberculosis
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the substrate capture mechanism of mycobacterium tuberculosis Anthranilate phosphoribosyltransferase provides a mode for inhibition
Biochemistry, 2013Co-Authors: Alina Castell, Esther M M Bulloch, Genevieve L. Evans, Emily J. Parker, T V M Cookson, Francesca L Short, Dmitri D A Joseph, Clare E Lee, Edward N. BakerAbstract:Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5′-phosphoribosyl 1′-pyrophosphate (PRPP) and Anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two Anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic Anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5–119 μM, with the strongest inhibition displayed by a biAnthranilate compound that makes hydrogen bond and sal...
Igor Jerković - One of the best experts on this subject based on the ideXlab platform.
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traceability of satsuma mandarin citrus unshiu marc honey through nectar honey sac honey pathways of the headspace volatiles and semi volatiles chemical markers
Molecules, 2016Co-Authors: Igor Jerković, Zvonimir Marijanovic, Marina Zekic, Lidija Svečnjak, Saša Prđun, Dragan Bubalo, Carlo Ignazio Giovanni TuberosoAbstract:Headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by GC-MS/FID, were applied for monitoring the nectar (NE)/honey-sac (HoS)/honey (HO) pathways of the headspace, volatiles, and semi-volatiles. The major NE (4 varieties of Citrus unshiu) headspace compounds were linalool, α-terpineol, 1H-indole, methyl Anthranilate, and phenylacetonitrile. Corresponding extracts contained, among others, 1H-indole, methyl Anthranilate, 1,3-dihydro-2H-indol-2-one and caffeine. The major HoS headspace compounds were linalool, α-terpineol, 1,8-cineole, 1H-indole, methyl Anthranilate, and cis-jasmone. Characteristic compounds from HoS extract were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl Anthranilate, and phenylacetonitrile. However, HO headspace composition was significantly different in comparison to NE and HoS with respect to phenylacetaldehyde and linalool derivatives abundance that appeared as the consequence of the hive conditions and the bee enzyme activity. C. unshiu honey traceability is determined by chemical markers: phenylacetaldehyde, phenylacetonitrile, linalool and its derivatives, as well as 1H-indole, 1,3-dihydro-2H-indol-2-one, and caffeine.
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Traceability of Satsuma Mandarin (Citrus unshiu Marc.) Honey through Nectar/Honey-Sac/Honey Pathways of the Headspace, Volatiles, and Semi-Volatiles: Chemical Markers
Molecules (Basel Switzerland), 2016Co-Authors: Igor Jerković, Lidija Svečnjak, Saša Prđun, Dragan Bubalo, Zvonimir Marijanović, Marina Zekić, Carlo Ignazio Giovanni TuberosoAbstract:Headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE), followed by GC-MS/FID, were applied for monitoring the nectar (NE)/honey-sac (HoS)/honey (HO) pathways of the headspace, volatiles, and semi-volatiles. The major NE (4 varieties of Citrus unshiu) headspace compounds were linalool, α-terpineol, 1H-indole, methyl Anthranilate, and phenylacetonitrile. Corresponding extracts contained, among others, 1H-indole, methyl Anthranilate, 1,3-dihydro-2H-indol-2-one and caffeine. The major HoS headspace compounds were linalool, α-terpineol, 1,8-cineole, 1H-indole, methyl Anthranilate, and cis-jasmone. Characteristic compounds from HoS extract were caffeine, 1H-indole, 1,3-dihydro-2H-indol-2-one, methyl Anthranilate, and phenylacetonitrile. However, HO headspace composition was significantly different in comparison to NE and HoS with respect to phenylacetaldehyde and linalool derivatives abundance that appeared as the consequence of the hive conditions and the bee enzyme activity. C. unshiu honey traceability is determined by chemical markers: phenylacetaldehyde, phenylacetonitrile, linalool and its derivatives, as well as 1H-indole, 1,3-dihydro-2H-indol-2-one, and caffeine.
Edward N. Baker - One of the best experts on this subject based on the ideXlab platform.
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The Substrate Capture Mechanism of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Provides a Mode for Inhibition
2016Co-Authors: Alina Castell, Esther M M Bulloch, Edward N. Baker, Emily J. Parker, Francesca L Short, Dmitri D A Joseph, Clare E Lee, Genevieve L. Evans, Tammie V. M. Cookson, Shaun J. LottAbstract:Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5′-phosphoribosyl 1′-pyrophosphate (PRPP) and Anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two Anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic Anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5–119 μM, with the strongest inhibition displayed by a biAnthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme’s activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action
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structures of mycobacterium tuberculosis Anthranilate phosphoribosyltransferase variants reveal the conformational changes that facilitate delivery of the substrate to the active site
Biochemistry, 2015Co-Authors: T V M Cookson, Genevieve L. Evans, Alina Castell, Edward N. Baker, J S Lott, Emily J. ParkerAbstract:Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and Anthranilate. The roles of residues predicted to be involved in Anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both Anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of Anthranilate to bind in three sites along an extended Anthranilate tunnel and expose the role of the mobile β2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The β2-α6 loop moves sequentially between a "folded" conformation, partially occluding the Anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows Anthranilate access via the tunnel to the active site. The return of the β2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind Anthranilate at the opening of the Anthranilate tunnel for subsequent reactions. Multiple Anthranilate molecules blocking the Anthranilate tunnel prevent the β2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme.
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Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site
2015Co-Authors: Tammie V. M. Cookson, Shaun J. Lott, Genevieve L. Evans, Alina Castell, Edward N. Baker, Emily J. ParkerAbstract:Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg2+-dependent reaction between 5′-phosphoribosyl-1′-pyrophosphate (PRPP) and Anthranilate. The roles of residues predicted to be involved in Anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both Anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of Anthranilate to bind in three sites along an extended Anthranilate tunnel and expose the role of the mobile β2−α6 loop in facilitating the enzyme’s sequential reaction mechanism. The β2−α6 loop moves sequentially between a “folded” conformation, partially occluding the Anthranilate tunnel, via an “open” position to a “closed” conformation, which supports PRPP binding and allows Anthranilate access via the tunnel to the active site. The return of the β2−α6 loop to the “folded” conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind Anthranilate at the opening of the Anthranilate tunnel for subsequent reactions. Multiple Anthranilate molecules blocking the Anthranilate tunnel prevent the β2−α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme
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alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by Anthranilate phosphoribosyltransferase from mycobacterium tuberculosis
Biochemical Journal, 2014Co-Authors: T V M Cookson, Esther M M Bulloch, Shaun J. Lott, Genevieve L. Evans, Alina Castell, Edward N. Baker, Francesca L Short, Emily J. ParkerAbstract:AnPRT (Anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis ( Mtb ). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5′-phosphoribosyl-1′-pyrophosphate) to Anthranilate to form PRA (5′-phosphoribosyl Anthranilate). Mtb -AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by Anthranilate was observed. Antimycobacterial fluoroAnthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb -AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with Anthranilate analogues reveal two distinct binding sites for Anthranilate. One site is located over 8 A (1 A=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site Anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for Anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner Anthranilate-binding site. It is proposed that Anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP. Abbreviations: AnPRT, Anthranilate phosphoribosyltransferase; Eco, Escherichia coli; FA, fluoroAnthranilate; F-PRA, fluoro-PRA; InGP, indole glycerol phosphate; InGPS, indole glycerol phosphate synthase; KIE, kinetic isotope effect; MA, methylAnthranilate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; M-PRA, methyl-PRA; Mtb, Mycobacterium tuberculosis; NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PR, 5′-phosphoribosyl; PRA, 5′-phosphoribosyl Anthranilate; PRAI, 5′-phosphoribosyl Anthranilate isomerase; PRPP, 5′-phosphoribosyl-1′-pyrophosphate; PRT, phosphoribosyltransferase; Sso, Sulfolobus solfataricus; TB, tuberculosis
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the substrate capture mechanism of mycobacterium tuberculosis Anthranilate phosphoribosyltransferase provides a mode for inhibition
Biochemistry, 2013Co-Authors: Alina Castell, Esther M M Bulloch, Genevieve L. Evans, Emily J. Parker, T V M Cookson, Francesca L Short, Dmitri D A Joseph, Clare E Lee, Edward N. BakerAbstract:Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5′-phosphoribosyl 1′-pyrophosphate (PRPP) and Anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two Anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic Anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5–119 μM, with the strongest inhibition displayed by a biAnthranilate compound that makes hydrogen bond and sal...