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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 reaction catalyzed by the precorrin reductase CobK in the context of the eight methyltransferase reactions leading from uroporphyrinogen III to hydrogenobyrinic acid which is subsequently converted to cobalamin (vitamin B12).Precorrin-6A is transformed into precorrin-6B the substrate for the methyltransferase CobL in a reaction dependent on the coenzyme NADPH.
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f1: The reaction catalyzed by the precorrin reductase CobK in the context of the eight methyltransferase reactions leading from uroporphyrinogen III to hydrogenobyrinic acid which is subsequently converted to cobalamin (vitamin B12).Precorrin-6A is transformed into precorrin-6B the substrate for the methyltransferase CobL in a reaction dependent on the coenzyme NADPH.

Mentions: Cobalamin (vitamin B12) is a cofactor for two enzymes, methylmalonyl-CoA mutase and methionine synthase, and is therefore an essential dietary requirement for humans. Around thirty biosynthetic steps are required to manufacture cobalamin in bacteria and archea, making cobalamin biosynthesis one of the most intricate in nature. We are interested in the synthesis of the corrin ring component, cobinamide, from the ubiquitous tetrapyrrole primogenitor uroporphyrinogen III by a series of reactions including eight S-adenosyl-L-methionine-dependent methylations, ring contraction and reduction12. Many of the intermediates in the pathway are labile and have only recently been identified in non-esterified form3. In this paper we investigate the structure of the precorrin-6A reductase CobK involved in the aerobic pathway (Fig. 1), there is a homologous enzyme CbiJ involved in the anaerobic pathway4 both enzymes catalyse the NADPH-dependent reduction of precorrin-6A5 to the dihydro derivative precorrin-6B6. Deuterium labelling and NMR spectroscopy demonstrated a hydride from NADPH is transferred to C19 of precorrin-6A before this intermediate is subsequently converted enzymatically into hydrogenobyrinic acid6.


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 reaction catalyzed by the precorrin reductase CobK in the context of the eight methyltransferase reactions leading from uroporphyrinogen III to hydrogenobyrinic acid which is subsequently converted to cobalamin (vitamin B12).Precorrin-6A is transformed into precorrin-6B the substrate for the methyltransferase CobL in a reaction dependent on the coenzyme NADPH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: The reaction catalyzed by the precorrin reductase CobK in the context of the eight methyltransferase reactions leading from uroporphyrinogen III to hydrogenobyrinic acid which is subsequently converted to cobalamin (vitamin B12).Precorrin-6A is transformed into precorrin-6B the substrate for the methyltransferase CobL in a reaction dependent on the coenzyme NADPH.
Mentions: Cobalamin (vitamin B12) is a cofactor for two enzymes, methylmalonyl-CoA mutase and methionine synthase, and is therefore an essential dietary requirement for humans. Around thirty biosynthetic steps are required to manufacture cobalamin in bacteria and archea, making cobalamin biosynthesis one of the most intricate in nature. We are interested in the synthesis of the corrin ring component, cobinamide, from the ubiquitous tetrapyrrole primogenitor uroporphyrinogen III by a series of reactions including eight S-adenosyl-L-methionine-dependent methylations, ring contraction and reduction12. Many of the intermediates in the pathway are labile and have only recently been identified in non-esterified form3. In this paper we investigate the structure of the precorrin-6A reductase CobK involved in the aerobic pathway (Fig. 1), there is a homologous enzyme CbiJ involved in the anaerobic pathway4 both enzymes catalyse the NADPH-dependent reduction of precorrin-6A5 to the dihydro derivative precorrin-6B6. Deuterium labelling and NMR spectroscopy demonstrated a hydride from NADPH is transferred to C19 of precorrin-6A before this intermediate is subsequently converted enzymatically into hydrogenobyrinic acid6.

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