Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy

Petrovich, Robert M.; Ruzicka, Frank J.; Reed, George H.; Frey, Perry A.

doi:10.1021/bi00159a019

Cited by 89 publications

(94 citation statements)

References 31 publications

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“…Analysis of the deduced amino acid sequence of SplB (10, 12) revealed three closely spaced cysteine residues (C91, C95, and C98) within a short region demonstrating a high degree of similarity to proteins belonging to the radical SAM superfamily (11,(25)(26)(27)(28). Radical SAM enzymes contain oxygen-labile [4Fe-4S] clusters that readily degrade to an oxidized [3Fe-4S] form; the oxidized and reduced forms generate distinct EPR spectra.…”

Section: Discussionmentioning

confidence: 99%

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

Rebeil

Nicholson

2001

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

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A n important property of bacterial endospores is their ability to survive high doses of UV radiation, which is due to the packaging of DNA and the unique photochemistry that this packaging imparts on dormant spores (1-3). The major photoproduct in DNA of UV-irradiated dormant spores is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally called spore photoproduct or SP (4, 5). SP can be removed from DNA during spore germination by the general nucleotide excision repair (NER) pathway (6, 7). In addition to NER, spores possess an SP-specific enzyme called SP lyase that can directly reverse SP to two thymines in situ without excision from DNA (6,8,9). Analysis of the deduced amino acid sequence encoded by the SP lyase gene (splB) cloned from Bacillus subtilis revealed a limited region with similarity to DNA photolyases (10), although early indirect experiments indicated that SP lyase caused direct reversal of SP back to two thymines in situ in a light-independent process (8, 9). These observations led to the question as to the mechanism SP lyase utilizes to reverse SP to two thymines. Inspection of the 342-aa deduced SplB sequence (10) revealed that the protein contains four cysteines, three of which are clustered at residues 91, 95, and 98, and the fourth at residue 141. The region from C91 to C98 was similar to the signature cysteine clusters present in the recently dubbed ''radical SAM'' protein superfamily (11), which includes enzymes such as anaerobic ribonucleotide reductase (RNR), pyruvate formate-lyase (PFL), lysine-2,3-aminomutase (KAM), and biotin synthase (BioB) (12). In radical SAM enzymes, the clustered cysteines serve as ligands in the formation of iron-sulfur ([Fe-S]) centers, and these enzymes use S-adenosylmethionine (S-AdoMet) as a cofactor to generate an adenosyl radical (13-16). We constructed a version of SplB protein containing an N-terminal tag of 10 histidines [(10His)SplB] and subsequently determined that (10His)SplB (i) contained both iron and acid-labile sulfur in a stoichiometric ratio of 1-2 atoms of iron or sulfur per (10His)SplB subunit (17); (ii) exhibited a UV-visible absorption spectrum consistent with other [Fe-S] proteins (17); (iii) required both anaerobic reducing conditions and S-AdoMet for activity in vitro (17, 18); and (iv) was inactive for SP cleavage unless its (10His) tag was first removed proteolytically (17, 18). The above evidence led us to the hypothesis that SP lyase carries out catalysis using an [Fe-S] center to cleave S-AdoMet, thus generating a 5Ј-adenosyl radical that could participate either directly or indirectly in SP cleavage. Support for this hypothesis has recently been obtained by Mehl and Begley (19). Using synthetic analogues of SP, they proposed a mechanistic scenario for SP reversal to two thymines in which the radical generated from S-AdoMet cleavage by electron donation from the [4Fe-4S] center can abstract a proton from C6Ј of SP, effectively generating an SP radical that fragments readily back to two thymines (Fig. 6) (19). The...

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Section: Discussionmentioning

confidence: 99%

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

Rebeil

Nicholson

2001

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

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“…Unlike other aminomutases, including D-lysine 5,6-aminomutase (1, 26), D-ornithine 2,3-aminomutase (2), and L-leucine 2,3-aminomutase (33), KAM is not adenosylcobalamin dependent. Instead, the enzyme is activated by SAM and contains iron-sulfur clusters and PLP (7,31,38).The interconversion of L-␣-lysine and L-␤-lysine is a reversible process in which an unactivated, carbon-bound hydrogen atom undergoes a 1,2-migration concomitant with the countermigration of the ␣-amino group by a novel free-radicalbased mechanism (12, 13). According to the current working hypothesis, homolytic cleavage of the S-adenosyl bond in SAM is mediated by the [4Fe-4S] center and produces the 5Ј-deoxyadenosyl radical as a transient intermediate (27,28).…”

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“…KAM is a multisubunit protein with a subunit M r of 48,000 and an overall M r of 285,000 (7,38). Later studies demonstrated the presence of labile sulfide in the form of three [4Fe-4S] centers (31) and six molecules of PLP per hexamer (38). Although KAM has been described as a hexamer of identical subunits, the amino acid composition and the precise molecular weight have not been established.…”

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Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

2000

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Lysine 2,3-aminomutase (KAM, EC 5.4.3.2.) catalyzes the interconversion of L-lysine and L-␤-lysine, the first step in lysine degradation in Clostridium subterminale SB4. KAM requires S-adenosylmethionine (SAM), which mediates hydrogen transfer in a mechanism analogous to adenosylcobalamin-dependent reactions. KAM also contains an iron-sulfur cluster and requires pyridoxal 5-phosphate (PLP) for activity. In the present work, we report the cloning and nucleotide sequencing of the gene kamA for C. subterminale SB4 KAM and conditions for its expression in Escherichia coli. The cyanogen bromide peptides were isolated and characterized by mass spectral analysis and, for selected peptides, amino acid and N-terminal amino acid sequence analysis. PCR was performed with degenerate oligonucleotide primers and C. subterminale SB4 chromosomal DNA to produce a portion of kamA containing 1,029 base pairs of the gene. KAM from Clostridium subterminale SB4 catalyzes the interconversion of L-lysine and L-␤-lysine. In C. subterminale SB4, the production of ␤-lysine is the first step in the metabolism of lysine as the sole source of carbon and nitrogen, leading to acetate and butyrate as final products (39). Unlike other aminomutases, including D-lysine 5,6-aminomutase (1, 26), D-ornithine 2,3-aminomutase (2), and L-leucine 2,3-aminomutase (33), KAM is not adenosylcobalamin dependent. Instead, the enzyme is activated by SAM and contains iron-sulfur clusters and PLP (7,31,38).The interconversion of L-␣-lysine and L-␤-lysine is a reversible process in which an unactivated, carbon-bound hydrogen atom undergoes a 1,2-migration concomitant with the countermigration of the ␣-amino group by a novel free-radicalbased mechanism (12, 13). According to the current working hypothesis, homolytic cleavage of the S-adenosyl bond in SAM is mediated by the [4Fe-4S] center and produces the 5Ј-deoxyadenosyl radical as a transient intermediate (27,28). As illustrated in Fig. 1, the 5Ј-deoxyadenosyl radical initiates the rearrangement by abstracting the 3-pro-R hydrogen of L-lysine in the forward direction to form radical 1 or the 2-pro-R hydrogen of L-␤-lysine in the reverse direction to form radical 3.Evidence suggests that both amino acids are bound to the enzyme in the form of external aldimines with PLP. The substrate-and product-related free radicals are interconverted by way of an azacyclopropylcarbinyl, radical intermediate 2.KAM was first observed by Costilow and coworkers in crude extracts of C. subterminale SB4 (8) and later was purified and found to contain PLP and iron and to require SAM for activity (7). Enzyme activity was rapidly destroyed by dioxygen. The purified enzyme was partially inactive and could be further activated by prolonged anaerobic incubation with PLP, iron, and glutathione or dihydrolipoate, followed by addition of SAM and dithionite (7). KAM is a multisubunit protein with a subunit M r of 48,000 and an overall M r of 285,000 (7,38). Later studies demonstrated the presence of labile sulfide in the form of three [4Fe-4S]...

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“…However, with improvements in purification and activation conditions, the enzyme proved to contain a [4Fe-4S] cluster. And optimization of the iron and sulfide content led to high activity, with a turnover number ~50 s -s , similar to the activities of adenosylcobalamin-dependent isomerases [12,13]. Therefore, SAM must have been at least as efficient as the Vitamin B 12 coenzyme.…”

Section: Letter To the Editormentioning

confidence: 99%

The Birth and Demise of Hypotheses on Evolution of S-Adenosyl-Lmethionine and Adenosylcobalamin

Frey¹

2017

Biochem Anal Biochem

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Relationships between adenosylcobalamin and S-adenosyl-Lmethionine (SAM)-dependent enzymatic radical reactions are explored with a view toward determining their evolutionary relationships. Adenosylcobalamin is a Vitamin B 12 -coenzyme, and the vitamin deficiency causes pernicious anemia in humans. Methionine, the precursor of SAM, is a nutritionally essential amino acid. Evidence implicates both SAM and adenosylcobalamin in the generation of the 5'-deoxyadenosyl radical as the initiator of carbon-centered radical chemistry. However, expectations of the evolutionary superiority of the structurally and chemically complex adenosylcobalamin as an initiator of radical biochemistry are contradicted by available information. It is pointed out that adenosylcobalamin functions equally well aerobically and anaerobically, whereas SAM requires strong reducing conditions and electron transfer mediated by a [4Fe-4S] 1+ cluster to initiate carboncentered radical chemistry. This paper focuses on the biochemical relationships between the Vitamin B 12 coenzyme adenosylcobalamin and S-adenosyl-L-methionine (SAM). Bacteria synthesize Vitamin B 12 (cobalamin), and bacteria and animals alkylate it to its coenzymatic forms adenosylcobalamin and methylcobalamin. A dietary deficiency of cobalamin leads to pernicious anemia in humans because of the importance of the B 12 -coenzymatic forms in metabolism. Adenosylcobalamin potentiates the interconversion of succinyl-CoA and methylmalonyl-CoA by methylmalonyl-CoA mutase [1], and methylcobalamin mediates methyltransfer from N 5

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Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy

Cited by 89 publications

References 31 publications

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase

Lysine 2,3-Aminomutase from Clostridium subterminale SB4: Mass Spectral Characterization of Cyanogen Bromide-Treated Peptides and Cloning, Sequencing, and Expression of the Gene kamA in Escherichia coli

The Birth and Demise of Hypotheses on Evolution of S-Adenosyl-Lmethionine and Adenosylcobalamin

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