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Cobalamin uptake and reactivation occurs through specific protein interactions in the methionine synthase-methionine synthase reductase complex.
FEBS J. 2009 Apr; 276(7):1942-51
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20 Apr 2009
Confirmation, New Finding, Technical Advance
DOI: 10.3410/f.1159916.620174
The authors have expanded our understanding of the incorporation of cobalamin into human methionine synthase, and demonstrated a complex that is formed between that enzyme and methionine synthase reductase. They provide convincing evidence for a specific role for methionine synthase reductase in cobalamin incorporation, but also show that many of the P450 reductase family members can catalyze aquocobalamin reduction to the cob(II)alamin form, which precedes its incorporation into methionine synthase.
Human methionine synthase has proved extremely difficult to express and purify and this paper reports its successful expression and purification from Pichia pastoris, making the in vitro studies with purified enzymes possible. The present study contributes significantly to our understanding of the complex processes by which extremely low concentrations of cobalamin are incorporated highly specifically into the two cobalamin-dependent enzymes in mammalian cells. Both of these enzymes bind cobalamin with its dimethylbenzimidazole substituent dissociated from the cobalt, the so-called base-off form of the cofactor. And critical to the formation of the base-off form is reduction of aquocobalamin to cob(II)alamin. Genetic and biochemical experiments in recent decades have elucidated the players in cobalamin trafficking, but curiously no aquocobalamin reductase was identified. The unexpected observation in this paper that other members of the P450 reductase family efficiently catalyze aquocobalamin reduction may explain the apparent lack of any specific reductases for the conversion of aquocobalamin to cob(II)alamin in mammalian cells.
Disclosures
None declared
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Matthews R: F1000Prime Recommendation of [Wolthers KR and Scrutton NS, FEBS J 2009, 276(7):1942-51]. In F1000Prime, 20 Apr 2009; DOI: 10.3410/f.1159916.620174. F1000Prime.com/1159916#eval620174
F1000Prime Recommendations, Dissents and Comments for [Wolthers KR and Scrutton NS, FEBS J 2009, 276(7):1942-51]. In F1000Prime, 19 Jun 2013; F1000Prime.com/1159916
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Human methionine synthase reductase (MSR), a diflavin enzyme, restores the activity of human methionine synthase through reductive methylation of methionine synthase (MS)-bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo-MS, a role associated with the MSR-catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA103, 9476-9481]. Here, we report the expression and purification of human methionine synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin reductase enzymes (cytochrome P450 reductase and the reductase domain of neuronal nitric oxide synthase) to: (a) stimulate formation of holo-MS from aquacob(III)alamin and the apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin reductases studied, cytochrome P450 reductase had the highest turnover rate (55.5 s(-1)) for aquacob(III)alamin reduction, and the reductase domain of neuronal nitric oxide synthase elicited the highest specificity (k(cat)/K(m) of 1.5 x 10(5) m(-1) s(-1)) and MSR had the lowest K(m) (6.6 microm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full-length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo-MS. MSR was also the only diflavin reductase to reactivate the inert cob(II)alamin form of purified human MS (K(act) of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo-MS is enhanced through specific protein-protein interactions between the MSR FMN domain and MS.
DOI: 10.1111/j.1742-4658.2009.06919.x
PMID: 19243433
