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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

The crystal structure of the precorrin reductase CobK.(a) Cartoon structure of CobK reveals two Rossmann-like folds facing each other across the NADPH and substrate binding cleft, the polypeptide chain is coloured from blue to orange from N- to C-terminus. The polypeptide chain crosses from the N-terminal domain to form the C-terminal before returning to complete the N-terminal domain. In essence, the final β-strands are swapped between domains. (b) The topology of CobK reveals the strand order in the two Rossmann-like fold domains. (c) Superimposition of the two domains (N- and C-terminal in cyan and green, respectively) reveals their topological similarity once the swapped β-stands are excluded (strand order 32145). (d) The polypeptide chain returning to the N-terminal domain forms a disulfide (Cys95–Cys231) with β5 and then forms the final helix, α8. This and the other figures of the structures were made using PyMOL, unless otherwise indicated.
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f2: The crystal structure of the precorrin reductase CobK.(a) Cartoon structure of CobK reveals two Rossmann-like folds facing each other across the NADPH and substrate binding cleft, the polypeptide chain is coloured from blue to orange from N- to C-terminus. The polypeptide chain crosses from the N-terminal domain to form the C-terminal before returning to complete the N-terminal domain. In essence, the final β-strands are swapped between domains. (b) The topology of CobK reveals the strand order in the two Rossmann-like fold domains. (c) Superimposition of the two domains (N- and C-terminal in cyan and green, respectively) reveals their topological similarity once the swapped β-stands are excluded (strand order 32145). (d) The polypeptide chain returning to the N-terminal domain forms a disulfide (Cys95–Cys231) with β5 and then forms the final helix, α8. This and the other figures of the structures were made using PyMOL, unless otherwise indicated.

Mentions: We have determined the structure of apoenzyme, holoenzyme and a ternary complex with NADPH and product giving unprecedented insights into the structure and mechanism of CobK. The description of the structure here will initially be based on the high-resolution structure of the ternary complex, and then differences between the structures of the ternary complex and the other structures will be described (Table 1 gives details of the resolution, data quality, and refinement). CobK unusually comprises two parallel β-sheet domains arranged so the β-strands point towards the centre of the molecule where the NADPH and substrate bind (Fig. 2). The structures reveal how coenzyme and substrate bind to the active centre and explain the specificity of CobK for precorrin-6A as well as elucidating the molecular basis of product-trapping.


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)

The crystal structure of the precorrin reductase CobK.(a) Cartoon structure of CobK reveals two Rossmann-like folds facing each other across the NADPH and substrate binding cleft, the polypeptide chain is coloured from blue to orange from N- to C-terminus. The polypeptide chain crosses from the N-terminal domain to form the C-terminal before returning to complete the N-terminal domain. In essence, the final β-strands are swapped between domains. (b) The topology of CobK reveals the strand order in the two Rossmann-like fold domains. (c) Superimposition of the two domains (N- and C-terminal in cyan and green, respectively) reveals their topological similarity once the swapped β-stands are excluded (strand order 32145). (d) The polypeptide chain returning to the N-terminal domain forms a disulfide (Cys95–Cys231) with β5 and then forms the final helix, α8. This and the other figures of the structures were made using PyMOL, unless otherwise indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The crystal structure of the precorrin reductase CobK.(a) Cartoon structure of CobK reveals two Rossmann-like folds facing each other across the NADPH and substrate binding cleft, the polypeptide chain is coloured from blue to orange from N- to C-terminus. The polypeptide chain crosses from the N-terminal domain to form the C-terminal before returning to complete the N-terminal domain. In essence, the final β-strands are swapped between domains. (b) The topology of CobK reveals the strand order in the two Rossmann-like fold domains. (c) Superimposition of the two domains (N- and C-terminal in cyan and green, respectively) reveals their topological similarity once the swapped β-stands are excluded (strand order 32145). (d) The polypeptide chain returning to the N-terminal domain forms a disulfide (Cys95–Cys231) with β5 and then forms the final helix, α8. This and the other figures of the structures were made using PyMOL, unless otherwise indicated.
Mentions: We have determined the structure of apoenzyme, holoenzyme and a ternary complex with NADPH and product giving unprecedented insights into the structure and mechanism of CobK. The description of the structure here will initially be based on the high-resolution structure of the ternary complex, and then differences between the structures of the ternary complex and the other structures will be described (Table 1 gives details of the resolution, data quality, and refinement). CobK unusually comprises two parallel β-sheet domains arranged so the β-strands point towards the centre of the molecule where the NADPH and substrate bind (Fig. 2). The structures reveal how coenzyme and substrate bind to the active centre and explain the specificity of CobK for precorrin-6A as well as elucidating the molecular basis of product-trapping.

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