Limits...
Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap.

Gu S, Sushko O, Deery E, Warren MJ, Pickersgill RW - Sci Rep (2015)

Bottom Line: CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway.The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK.The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.

View Article: PubMed Central - PubMed

Affiliation: Chemistry &Biochemistry Department, School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK.

ABSTRACT
CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel β-sheets have swapped their last β-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold. The trapped ternary complex with coenzyme and product reveals five conserved basic residues that bind the carboxylates of the tetrapyrrole tightly anchoring the product. A loop, disordered in both the apoenzyme and holoenzyme structures, closes around the product further tightening binding. The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK. The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.

No MeSH data available.


Related in: MedlinePlus

Product binding in detail.(a) NADPH and precorrin drawn in stick representation showing their relative orientation within the active centre of CobK. The geometry and separation of nicotinamide C4N and precorrin C19, 2.8 Å, is consistent with hydride transfer from C4N to C19. The carbons of the nicotinamide and precorrin are shown in magenta and cyan respectively. Oxygen atoms are red and nitrogen atoms blue; the small red spheres are water molecules shown as the proton may be provided by water to C18. (b) In silico the product precorrin-6B (magenta carbons) docks is a closely similar orientation to the bound product seen in the experimental structure (green carbons). (c) In silico the substrate precorrin-6A (yellow carbons) also docks into the active centre of CobK in similar orientation to that observed experimentally and clearly shows the changes in conformation that accompanies reduction of the C18-C19 bond. (d) Surface representation of the ternary complex. CobK surface coloured according to conservation in magenta (highly conserved) to cyan (poorly conserved), precorrin surface in orange, and NADPH surface in blue. The figure highlights the almost complete burial of the precorrin, this is the only surface of the precorrin visible in any orientation of the molecule and only the proprionate of ring-B can be seen protruding close to the conserved sequences that bind the substrate and product. This panel was produced using Chimera20.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663756&req=5

f4: Product binding in detail.(a) NADPH and precorrin drawn in stick representation showing their relative orientation within the active centre of CobK. The geometry and separation of nicotinamide C4N and precorrin C19, 2.8 Å, is consistent with hydride transfer from C4N to C19. The carbons of the nicotinamide and precorrin are shown in magenta and cyan respectively. Oxygen atoms are red and nitrogen atoms blue; the small red spheres are water molecules shown as the proton may be provided by water to C18. (b) In silico the product precorrin-6B (magenta carbons) docks is a closely similar orientation to the bound product seen in the experimental structure (green carbons). (c) In silico the substrate precorrin-6A (yellow carbons) also docks into the active centre of CobK in similar orientation to that observed experimentally and clearly shows the changes in conformation that accompanies reduction of the C18-C19 bond. (d) Surface representation of the ternary complex. CobK surface coloured according to conservation in magenta (highly conserved) to cyan (poorly conserved), precorrin surface in orange, and NADPH surface in blue. The figure highlights the almost complete burial of the precorrin, this is the only surface of the precorrin visible in any orientation of the molecule and only the proprionate of ring-B can be seen protruding close to the conserved sequences that bind the substrate and product. This panel was produced using Chimera20.

Mentions: The electron density of the ternary complex clearly reveals that it is product that is bound rather than substrate as both C18 and C19 are sp3 hybridized (Fig. 3c shows the clarity of the map). The product bound to CobK has been further modified by methylation of C5, this is a modification that can be accommodated within the active centre of the enzyme and points to the lability of the intermediate through the crystallization and data collection steps. The origin of this methyl-group is unclear. Four loops from the substrate-binding domain contribute to the substrate-binding site: β7/α5, β8/β9, β9/α6 and β10/α7. Three loops from the coenzyme-binding domain: β1/α1, β2/β3 and β4/α2 also contribute to the binding-site. The extensive network of interactions that bind the precorrin to the enzyme include five conserved basic residues (Fig. 3d). Sequence conservation is shown in Supplementary Figure S3. At the active centre of CobK the coenzyme and product are held with geometry and relative orientation consistent with the hydride having been transferred from C4 of the nicotinamide to C19 of the precorrin-6B product (Fig. 4a).


Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap.

Gu S, Sushko O, Deery E, Warren MJ, Pickersgill RW - Sci Rep (2015)

Product binding in detail.(a) NADPH and precorrin drawn in stick representation showing their relative orientation within the active centre of CobK. The geometry and separation of nicotinamide C4N and precorrin C19, 2.8 Å, is consistent with hydride transfer from C4N to C19. The carbons of the nicotinamide and precorrin are shown in magenta and cyan respectively. Oxygen atoms are red and nitrogen atoms blue; the small red spheres are water molecules shown as the proton may be provided by water to C18. (b) In silico the product precorrin-6B (magenta carbons) docks is a closely similar orientation to the bound product seen in the experimental structure (green carbons). (c) In silico the substrate precorrin-6A (yellow carbons) also docks into the active centre of CobK in similar orientation to that observed experimentally and clearly shows the changes in conformation that accompanies reduction of the C18-C19 bond. (d) Surface representation of the ternary complex. CobK surface coloured according to conservation in magenta (highly conserved) to cyan (poorly conserved), precorrin surface in orange, and NADPH surface in blue. The figure highlights the almost complete burial of the precorrin, this is the only surface of the precorrin visible in any orientation of the molecule and only the proprionate of ring-B can be seen protruding close to the conserved sequences that bind the substrate and product. This panel was produced using Chimera20.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663756&req=5

f4: Product binding in detail.(a) NADPH and precorrin drawn in stick representation showing their relative orientation within the active centre of CobK. The geometry and separation of nicotinamide C4N and precorrin C19, 2.8 Å, is consistent with hydride transfer from C4N to C19. The carbons of the nicotinamide and precorrin are shown in magenta and cyan respectively. Oxygen atoms are red and nitrogen atoms blue; the small red spheres are water molecules shown as the proton may be provided by water to C18. (b) In silico the product precorrin-6B (magenta carbons) docks is a closely similar orientation to the bound product seen in the experimental structure (green carbons). (c) In silico the substrate precorrin-6A (yellow carbons) also docks into the active centre of CobK in similar orientation to that observed experimentally and clearly shows the changes in conformation that accompanies reduction of the C18-C19 bond. (d) Surface representation of the ternary complex. CobK surface coloured according to conservation in magenta (highly conserved) to cyan (poorly conserved), precorrin surface in orange, and NADPH surface in blue. The figure highlights the almost complete burial of the precorrin, this is the only surface of the precorrin visible in any orientation of the molecule and only the proprionate of ring-B can be seen protruding close to the conserved sequences that bind the substrate and product. This panel was produced using Chimera20.
Mentions: The electron density of the ternary complex clearly reveals that it is product that is bound rather than substrate as both C18 and C19 are sp3 hybridized (Fig. 3c shows the clarity of the map). The product bound to CobK has been further modified by methylation of C5, this is a modification that can be accommodated within the active centre of the enzyme and points to the lability of the intermediate through the crystallization and data collection steps. The origin of this methyl-group is unclear. Four loops from the substrate-binding domain contribute to the substrate-binding site: β7/α5, β8/β9, β9/α6 and β10/α7. Three loops from the coenzyme-binding domain: β1/α1, β2/β3 and β4/α2 also contribute to the binding-site. The extensive network of interactions that bind the precorrin to the enzyme include five conserved basic residues (Fig. 3d). Sequence conservation is shown in Supplementary Figure S3. At the active centre of CobK the coenzyme and product are held with geometry and relative orientation consistent with the hydride having been transferred from C4 of the nicotinamide to C19 of the precorrin-6B product (Fig. 4a).

Bottom Line: CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway.The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK.The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.

View Article: PubMed Central - PubMed

Affiliation: Chemistry &Biochemistry Department, School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London, E1 4NS, UK.

ABSTRACT
CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel β-sheets have swapped their last β-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold. The trapped ternary complex with coenzyme and product reveals five conserved basic residues that bind the carboxylates of the tetrapyrrole tightly anchoring the product. A loop, disordered in both the apoenzyme and holoenzyme structures, closes around the product further tightening binding. The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK. The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.

No MeSH data available.


Related in: MedlinePlus