Methionine Biosynthesis in Enterobacteriaceae: Biochemical, Regulatory, and Evolutionary Aspect

Saint‐Girons, Isabelle; Parsot, Claude; Mm, Zakin; Bârzu, Octavian; Gn, Cohen

doi:10.3109/10409238809083374

Cited by 100 publications

(83 citation statements)

References 156 publications

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“…Minimally, we find that biotin synthase employs the last mechanism to reduce and or slow binding of AdoMet in the absence of DTB. At physiologic AdoMet concentrations [∼80 μM in E. coli (39)], biotin synthase binds weakly to 1 equiv of AdoMet [K d (exp) = 100 μM ( Figure 1A), K dl+2 (calcd) = 90 μM] and probably binds with comparable affinity but much slower kinetics to a second equivalent of AdoMet per dimer [k on = 10 M -1 s -1 ( Figure 9)]. However, in the presence of DTB, biotin synthase binds 2 equiv of AdoMet with high affinity [apparent K d = 1-5 μM ( Figures 1A and 2B)].…”

Section: Discussionmentioning

confidence: 99%

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

2003

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Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5′-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S] 2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S] 2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S] 2+ cluster and the [4Fe-4S] 2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at ∼400 nm that we attribute to changes in the coordination environment of the [4Fe-4S] 2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5′-deoxyadenosyl radicals in the absence of the correct substrate.Biotin synthase (BS) 1 catalyzes formation of the thioether ring in the essential vitamin biotin (for reviews, see refs 1 and 2). Thioether ring formation requires substitution of sulfur for hydrogen atoms at the saturated C6 and C9 positions of the substrate dethiobiotin. Functionalization of these relatively unreactive carbon atoms in dethiobiotin is likely initiated by hydrogen atom abstraction (3), generating carbon radicals at one or both positions. Biotin synthase is purified from Escherichia coli as a dimeric iron-sulfur protein (4) that requires S- † This research has been supported by NIH Research Grant GM59175 (J.T.J.).* To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 905B Stellar-Chance Laboratories, Philadelphia, PA 19104-6059...

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Section: Discussionmentioning

confidence: 99%

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

2003

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“…The final reaction in the pathway is the transfer of a methyl group to homocysteine (for a review see (60)). Methylation of homocysteine can be performed by two transmethylases (61,62). The metE gene product is a B12-independent enzyme and the metes gene product is a B12-dependent transmethylase.…”

Section: Cobalamins (Vitamin B12) Prokaryotesmentioning

confidence: 99%

“…It is negatively controlled by the metJ gene product with S-adenosylmethionine (AdoMet) acting as a corepressor (61)(62)(63)(64). The metE gene is also repressed by the addition of B12 to the growth medium (65).…”

Section: Cobalamins (Vitamin B12) Prokaryotesmentioning

confidence: 99%

Regulation of Gene Expression by Cofactors Derived from B Vitamins.

Brandsch

1994

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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SummaryVitamins represent precursor molecules for the majority of cofactors essential for various enzyme activities. Is the expression of proteins that require a specific cofactor for their function correlated to its availability? Examples are presented and discussed of the involvement of vitamin-derived cofactors in the regulatory mechanisms controlling gene expression in bacteria and eukaryotes at the transcriptional and trans lational level.

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“…The metE gene, as well as all of the other methionine biosynthetic genes except metH, is negatively controlled by the metJ gene product, with S-adenosylmethionine (SAM) acting as the corepressor (14,15). In addition, the metE gene is repressed by the addition of B12 to the growth medium (7,8).…”

mentioning

confidence: 99%

“…In Escherichia coli and Salmonella typhimurium, the final reaction in the synthesis of methionine is the methylation of homocysteine (14,15) (Fig. 1).…”

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confidence: 99%

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

Urbanowski

Stauffer

1992

J Bacteriol

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The vitamin B12 (B.2)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a A phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-Bl2 holoenzyme.

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Methionine Biosynthesis in Enterobacteriaceae: Biochemical, Regulatory, and Evolutionary Aspect

Cited by 100 publications

References 156 publications

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

Regulation of Gene Expression by Cofactors Derived from B Vitamins.

Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene

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