Interaction of Flavodoxin with Cobalamin-Dependent Methionine Synthase

Hall, D A; Jordan-Starck, Tuajuanda C.; Loo, Rachel Ogorzalek; Ludwig, Martha; Matthews, Rowena G.

doi:10.1021/bi001096c

Cited by 50 publications

(62 citation statements)

References 33 publications

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“…This residue has previously been shown to form a covalent crosslink with Lys-959 of this methionine synthase module (25). Glu-61 is located at the periphery of the observed surface (Fig.…”

Section: Resultsmentioning

confidence: 78%

“…To identify the surfaces on flavodoxin that bind its physiological partners, we performed 1 H, 15 N-HSQC NMR experiments in which we titrated 15 N, 2 H-labeled flavodoxin (20 kDa) with flavodoxin reductase (28 kDa) and, separately, with the AdoMet-binding region of methionine synthase (38 kDa). The latter module (methionine synthase residues 897-1227) was used in lieu of the entire 136-kDa methionine synthase protein because it is required for reductive activation (24) and because it was previously shown by crosslinking experiments to contain determinants for binding to flavodoxin (25). Only the peptide backbone and amide-containing side chains of flavodoxin were observed in the HSQC titrations, because flavodoxin reductase and the AdoMet-binding region of methionine synthase were not 15 N-labeled.…”

Section: Resultsmentioning

confidence: 99%

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Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase

Hall

Kooi

Stasik

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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102

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Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.

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“…This residue has previously been shown to form a covalent crosslink with Lys-959 of this methionine synthase module (25). Glu-61 is located at the periphery of the observed surface (Fig.…”

Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase

Hall

Kooi

Stasik

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

102

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“…The A66G and C524T sequence variants change the coding sequence from isoleucine to methionine (Ile22Met) and from serine to leucine (Ser175Leu), respectively. The A66G variant is located in the FMN domain and may therefore interact with methionine synthase (Hall et al, 2000;. Although we did not measure plasma Hcy levels, the 66G/524C haplotype was found to have 4-fold lower enzyme activity than wild type (Olteanu et al, 2002) and the 66G allele has Table 3.…”

Section: Discussionmentioning

confidence: 87%

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

Kim

Park²,

Koh³

et al. 2006

Exp Mol Med

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Homocysteine (Hcy) is thought to play an important role in the development of osteoporosis and fracture. Methionine synthase reductase (MTRR) is an enzyme involved in the conversion of Hcy to methionine. We hypothesized that certain genetic polymorphisms of MTRR leading to reduced enzyme activity may cause hyperhomocysteinemia and affect bone metabolism. We therefore examined the associations of the A66G and C524T polymorphisms of the MTRR gene with bone mineral density (BMD) and serum osteocalcin levels in postmenopausal women. Although we did not detect any significant associations between MTRR polymorphisms and BMD or serum osteocalcin levels, we found that the 66G/524C haplotype, which has reduced enzyme activity, was significantly associated with serum osteocalcin levels in a gene-dose dependent manner (P = 0.002). That is, the highest osteocalcin levels (34.5 ± 16.8 ng/ml) were observed in subjects bearing two copies, intermediate osteocalcin levels (32.6 ± 14.4 ng/ml) were observed in subjects bearing one copy, and the lowest levels of osteocalcin (28.8 ± 10.9 ng/ml) were observed in subjects bearing no copies. These results suggest that the 66G/524C haplotype of the MTRR gene affect bone turn over rate.

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“…Recombinant wild-type E. coli MS, flavodoxin, and His-tagged FNR were prepared according to the methods of Jarrett et al (32) and Hall et al (7).…”

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confidence: 99%

Human methionine synthase reductase is a molecular chaperone for human methionine synthase

Yamada

Gravel

Toraya

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K act value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37°C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS.aquacobalamin reductase ͉ cobalamin ͉ holoenzyme formation

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Interaction of Flavodoxin with Cobalamin-Dependent Methionine Synthase

Cited by 50 publications

References 33 publications

Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase

Mapping the interactions between flavodoxin and its physiological partners flavodoxin reductase and cobalamin-dependent methionine synthase

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

Human methionine synthase reductase is a molecular chaperone for human methionine synthase

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