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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.


The crystal structure of the ternary complex.(a) The binding sites of coenzyme NADPH and precorrin product relative to the domain architecture of CobK with β2/β3 loop (residues 33 to 40) that becomes ordered on product-binding indicated. (b) In the holoenzyme structure the nicotinamide phosphate and atoms to the nicotinamide side of the coenzyme are not seen, but in the ternary complex Arg34 makes hydrogen bonds to the nicotinamide phosphate (NP) and ribose ring oxygen and the coenzyme is much better defined. Adenosine binds on the surface of the β-sheet with the 2’-phosphate binding amides of residues 50, 51 and 52. Phe50 and Met79 contribute to the hydrophobic pocket binding the adenosine. The adenosine phosphate (AP) binds main chain amides of residues 78 and 79 at the N-terminal end of helix α3, but beyond the nicotinamide phosphate (NP) the coenzyme conformation is poorly defined in the holoenzyme structure and is presumed to be mobile. (c) Electron density corresponding to the precorrin product showing great clarity and enabling the contacts between enzyme and product to be confidently assigned. This σA-weighted 2Fobs-Fcalc map is contoured at 1.5σ. (d) Charged residues that interact with the precorrin product. These are conserved basic residues belonging to the C-terminal domain. The NADPH has been removed from the top of the product in this panel to reveal the interactions with charged residues. Note that the precorrin orientation is the same in Figures: 1; 3c; 3d; 4b and 4c with the rings labelled in Fig. 3d. LIGPLOT figures of the ligand interactions and the sequence alignment are shown as supplementary Figures S1, S2 and S3, respectively.
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f3: The crystal structure of the ternary complex.(a) The binding sites of coenzyme NADPH and precorrin product relative to the domain architecture of CobK with β2/β3 loop (residues 33 to 40) that becomes ordered on product-binding indicated. (b) In the holoenzyme structure the nicotinamide phosphate and atoms to the nicotinamide side of the coenzyme are not seen, but in the ternary complex Arg34 makes hydrogen bonds to the nicotinamide phosphate (NP) and ribose ring oxygen and the coenzyme is much better defined. Adenosine binds on the surface of the β-sheet with the 2’-phosphate binding amides of residues 50, 51 and 52. Phe50 and Met79 contribute to the hydrophobic pocket binding the adenosine. The adenosine phosphate (AP) binds main chain amides of residues 78 and 79 at the N-terminal end of helix α3, but beyond the nicotinamide phosphate (NP) the coenzyme conformation is poorly defined in the holoenzyme structure and is presumed to be mobile. (c) Electron density corresponding to the precorrin product showing great clarity and enabling the contacts between enzyme and product to be confidently assigned. This σA-weighted 2Fobs-Fcalc map is contoured at 1.5σ. (d) Charged residues that interact with the precorrin product. These are conserved basic residues belonging to the C-terminal domain. The NADPH has been removed from the top of the product in this panel to reveal the interactions with charged residues. Note that the precorrin orientation is the same in Figures: 1; 3c; 3d; 4b and 4c with the rings labelled in Fig. 3d. LIGPLOT figures of the ligand interactions and the sequence alignment are shown as supplementary Figures S1, S2 and S3, respectively.

Mentions: Coenzyme binds between the two domains making contacts with the N-terminal, coenzyme-binding domain (Fig. 3a). The sides of the adenosine–binding pocket is formed on one side by the β3/α2 loop and on the other by the β4/α3 loop α3. The floor of the pocket is provided by the surface of the parallel β-strands β1, β2, β3, β4 (Fig. 3a). In the holoenzyme the β2/β3 is disordered and the nicotinamide of the NADPH is not clearly defined. Large hydrophobic residues forming the adenosine-binding site are F50 and M79 from the β3/α2 loop and the N-terminus of helix α3, respectively. The 2’-phosphate binding site of NADPH is formed by main-chain amides of residues Phe50, Gly51 and Gly52 of the glycine-rich β3/α2-loop and by the NH2 of Asn82 from helix α2. The pyrophosphate-binding region of the coenzyme-binding domain has no glycine-rich fingerprint typical of NAD(P)-binding Rossmann folds. Contacts between NADPH and enzyme are presented in Supplementary Figure S1 and Supplementary Table S1. There are glycine residues in the polypeptide chain preceding α2 and α3 but no typical fingerprint and as a consequence unlike the typical situation in alcohol dehydrogenase where both the A and N phosphates are close to the N-terminal end of the pyrophosphate-binding helix (and helix-dipole), only the A phosphate is close to the N-terminal end of α2 and makes hydrogen-bonds the helix amides. The A phosphate group is slightly off-axis and not experiencing the full beneficial effect of the helix-dipole. Although the density is good for adenosine moiety the density falls away rapidly after the nicotinamide phosphate because the N phosphate, nicotinamide ribose and nicotinamide make few contacts with the enzyme and are not in a fixed conformation in the holoenzyme.


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 ternary complex.(a) The binding sites of coenzyme NADPH and precorrin product relative to the domain architecture of CobK with β2/β3 loop (residues 33 to 40) that becomes ordered on product-binding indicated. (b) In the holoenzyme structure the nicotinamide phosphate and atoms to the nicotinamide side of the coenzyme are not seen, but in the ternary complex Arg34 makes hydrogen bonds to the nicotinamide phosphate (NP) and ribose ring oxygen and the coenzyme is much better defined. Adenosine binds on the surface of the β-sheet with the 2’-phosphate binding amides of residues 50, 51 and 52. Phe50 and Met79 contribute to the hydrophobic pocket binding the adenosine. The adenosine phosphate (AP) binds main chain amides of residues 78 and 79 at the N-terminal end of helix α3, but beyond the nicotinamide phosphate (NP) the coenzyme conformation is poorly defined in the holoenzyme structure and is presumed to be mobile. (c) Electron density corresponding to the precorrin product showing great clarity and enabling the contacts between enzyme and product to be confidently assigned. This σA-weighted 2Fobs-Fcalc map is contoured at 1.5σ. (d) Charged residues that interact with the precorrin product. These are conserved basic residues belonging to the C-terminal domain. The NADPH has been removed from the top of the product in this panel to reveal the interactions with charged residues. Note that the precorrin orientation is the same in Figures: 1; 3c; 3d; 4b and 4c with the rings labelled in Fig. 3d. LIGPLOT figures of the ligand interactions and the sequence alignment are shown as supplementary Figures S1, S2 and S3, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: The crystal structure of the ternary complex.(a) The binding sites of coenzyme NADPH and precorrin product relative to the domain architecture of CobK with β2/β3 loop (residues 33 to 40) that becomes ordered on product-binding indicated. (b) In the holoenzyme structure the nicotinamide phosphate and atoms to the nicotinamide side of the coenzyme are not seen, but in the ternary complex Arg34 makes hydrogen bonds to the nicotinamide phosphate (NP) and ribose ring oxygen and the coenzyme is much better defined. Adenosine binds on the surface of the β-sheet with the 2’-phosphate binding amides of residues 50, 51 and 52. Phe50 and Met79 contribute to the hydrophobic pocket binding the adenosine. The adenosine phosphate (AP) binds main chain amides of residues 78 and 79 at the N-terminal end of helix α3, but beyond the nicotinamide phosphate (NP) the coenzyme conformation is poorly defined in the holoenzyme structure and is presumed to be mobile. (c) Electron density corresponding to the precorrin product showing great clarity and enabling the contacts between enzyme and product to be confidently assigned. This σA-weighted 2Fobs-Fcalc map is contoured at 1.5σ. (d) Charged residues that interact with the precorrin product. These are conserved basic residues belonging to the C-terminal domain. The NADPH has been removed from the top of the product in this panel to reveal the interactions with charged residues. Note that the precorrin orientation is the same in Figures: 1; 3c; 3d; 4b and 4c with the rings labelled in Fig. 3d. LIGPLOT figures of the ligand interactions and the sequence alignment are shown as supplementary Figures S1, S2 and S3, respectively.
Mentions: Coenzyme binds between the two domains making contacts with the N-terminal, coenzyme-binding domain (Fig. 3a). The sides of the adenosine–binding pocket is formed on one side by the β3/α2 loop and on the other by the β4/α3 loop α3. The floor of the pocket is provided by the surface of the parallel β-strands β1, β2, β3, β4 (Fig. 3a). In the holoenzyme the β2/β3 is disordered and the nicotinamide of the NADPH is not clearly defined. Large hydrophobic residues forming the adenosine-binding site are F50 and M79 from the β3/α2 loop and the N-terminus of helix α3, respectively. The 2’-phosphate binding site of NADPH is formed by main-chain amides of residues Phe50, Gly51 and Gly52 of the glycine-rich β3/α2-loop and by the NH2 of Asn82 from helix α2. The pyrophosphate-binding region of the coenzyme-binding domain has no glycine-rich fingerprint typical of NAD(P)-binding Rossmann folds. Contacts between NADPH and enzyme are presented in Supplementary Figure S1 and Supplementary Table S1. There are glycine residues in the polypeptide chain preceding α2 and α3 but no typical fingerprint and as a consequence unlike the typical situation in alcohol dehydrogenase where both the A and N phosphates are close to the N-terminal end of the pyrophosphate-binding helix (and helix-dipole), only the A phosphate is close to the N-terminal end of α2 and makes hydrogen-bonds the helix amides. The A phosphate group is slightly off-axis and not experiencing the full beneficial effect of the helix-dipole. Although the density is good for adenosine moiety the density falls away rapidly after the nicotinamide phosphate because the N phosphate, nicotinamide ribose and nicotinamide make few contacts with the enzyme and are not in a fixed conformation in the holoenzyme.

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.