Journal:Acta Cryst F:S2053230X25006181
From Proteopedia
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<scene name='10/1087727/021_Fig_3b_No_Lab_pse/2'>021 Fig 3b Cut Label 6x_pse</scene> | <scene name='10/1087727/021_Fig_3b_No_Lab_pse/2'>021 Fig 3b Cut Label 6x_pse</scene> | ||
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| + | Bacteria contain either one of two pathways to make menaquinone, the classical or futalosine pathways, and MenD catalyses the first irreversible step in the classical pathway. It takes two substrates, 2-oxoglutarate and isochorismate and converts them to SEPHCHC via a series of reactions involving covalent ThDP-bound intermediates. We have solved several structures of MenD from the listeria-causing pathogen Listeria monocytogenes. Analysis of the structures show a typical <scene name='10/1087727/021_fig_2a_png/5'>ThDP-dependent three-domain</scene> (PP, PYR, TH3 domain) fold similar to other MenD enzymes. Our <scene name='10/1087727/021_Fig_3a_No_Lab_pse/3'>first structure, captured in the ThDP cofactor-bound form</scene> has enabled us to visualise and understand how the co-factor binds. By comparing this structure to one without ThDP-bound (apo) in the protein databank we can see how the enzyme active site partially-closes around the co-factor. In our second structure we were able to capture reactivity within the crystal with an <scene name='10/1087727/021_Fig_3b_No_Lab_pse/3'>in-crystallo formed covalent ThDP-intermediate (Intermediate I) bound in the active site</scene>. Studying the shape and interactions of this intermediate helps us understand more about the chemistry of the enzyme <jmolButton> | ||
| + | <text>animation between 1st and 2nd determined structures</text> | ||
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var a = [1,2,3,4,5]; | var a = [1,2,3,4,5]; | ||
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| - | Bacteria contain either one of two pathways to make menaquinone, the classical or futalosine pathways, and MenD catalyses the first irreversible step in the classical pathway. It takes two substrates, 2-oxoglutarate and isochorismate and converts them to SEPHCHC via a series of reactions involving covalent ThDP-bound intermediates. We have solved several structures of MenD from the listeria-causing pathogen Listeria monocytogenes. Analysis of the structures show a typical <scene name='10/1087727/021_fig_2a_png/5'>ThDP-dependent three-domain</scene> (PP, PYR, TH3 domain) fold similar to other MenD enzymes. Our <scene name='10/1087727/021_Fig_3a_No_Lab_pse/3'>first structure, captured in the ThDP cofactor-bound form</scene> has enabled us to visualise and understand how the co-factor binds. By comparing this structure to one without ThDP-bound (apo) in the protein databank we can see how the enzyme active site partially-closes around the co-factor. In our second structure we were able to capture reactivity within the crystal with an <scene name='10/1087727/021_Fig_3b_No_Lab_pse/3'>in-crystallo formed covalent ThDP-intermediate (Intermediate I) bound in the active site</scene>. Studying the shape and interactions of this intermediate helps us understand more about the chemistry of the enzyme. | ||
In additional studies we used our structural analysis combined with other methods (size-exclusion chromatography, mass photometry and small angle X-ray scattering analysis) to understand the oligomeric state of the enzyme. Showing that like other MenD’s, Listeria monocytogenes MenD has a homotetrameric quaternary structure. We also undertook enzyme kinetics to show the enzyme was active, and that there was weak inhibition of enzyme activity in the presence of 1,4- dihydroxy-2-naphthoic acid, a downstream metabolite in the menaquinone-biosynthesis pathway that has previously been shown to be a potent allosteric regulator of Mycobacterium tuberculosis MenD. | In additional studies we used our structural analysis combined with other methods (size-exclusion chromatography, mass photometry and small angle X-ray scattering analysis) to understand the oligomeric state of the enzyme. Showing that like other MenD’s, Listeria monocytogenes MenD has a homotetrameric quaternary structure. We also undertook enzyme kinetics to show the enzyme was active, and that there was weak inhibition of enzyme activity in the presence of 1,4- dihydroxy-2-naphthoic acid, a downstream metabolite in the menaquinone-biosynthesis pathway that has previously been shown to be a potent allosteric regulator of Mycobacterium tuberculosis MenD. | ||
Revision as of 12:44, 25 August 2025
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This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
