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Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase.

Lanz ND, Pandelia ME, Kakar ES, Lee KH, Krebs C, Booker SJ - Biochemistry (2014)

Bottom Line: LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA(•)).Generation of the cross-linked species with a small, unlabeled (N(6)-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction.Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S](0) clusters.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and ‡Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.

ABSTRACT
Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N(6)-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA(•)). In the LS reaction, two equivalents of 5'-dA(•) are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe-4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N(6)-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S](0) clusters.

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SDS-PAGE analysis offractions from DE-52 column. Lanes are molecularweight markers (M), flow-through during column load (A and B), andsamples from eluted 10 mL fractions as the NaCl gradient was increasedfrom 0.1–1 M (1–14). Fractions that were pooled andused in further experiments are boxed.
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fig2: SDS-PAGE analysis offractions from DE-52 column. Lanes are molecularweight markers (M), flow-through during column load (A and B), andsamples from eluted 10 mL fractions as the NaCl gradient was increasedfrom 0.1–1 M (1–14). Fractions that were pooled andused in further experiments are boxed.

Mentions: To understand the natureof the turnover-dependent interaction between LS and its substrateand to provide evidence for a potential intermediate in catalysis,we studied the Ec LS reaction with [8,8,8-2H3]-OHP. Because sulfur insertion takes place at C6 beforeC8 and because quantitative deuterium substitution at C8 of the octanoylmoiety results essentially in arrest of turnover after the first sulfurinsertion, our working model would predict that OHP and LS shouldbecome cross-linked through the auxiliary cluster under turnover conditionswhen using [8,8,8-2H3]-OHP as substrate. Ec HP binds tightly to Ec LS; however,the two proteins can be separated by anion-exchange chromatography. Ec HP (pI = 4.59) adheres strongly to anion-exchange resinunder conditions described in Materials and Methods, whereas Ec LS (pI = 8.15) adheres poorly, if atall, under the same conditions. Figure 2A showsan SDS-PAGE analysis of fractions eluting from a Mono Q column underincreasing concentrations of NaCl. Ec LS and [8,8,8-2H3]-OHP were applied to the column after theircoincubation under turnover conditions in the absence of requiredlow-potential reductant. LS elutes essentially in the void volumeof the column (fractions 1–6), whereas [8,8,8-2H3]-OHP begins to elute at approximately 750 mM NaCl. The faintband that migrates slightly higher than [8,8,8-2H3]-OHP corresponds to LHP (or monothiolated LHP), which sometimescontaminates our preparations of apo-HP that are used to prepare OHP.When the proteins are incubated both in the presence of SAM and requiredreductant (sodium dithionite), a new species appears that displaysmigratory properties that are intermediate between those of LS andOHP (Figure 2B; fractions 20–25). Forreasons that we do not understand, OHP supports no more than 30–40%turnover (i.e., 0.3–0.4 equiv LHP per LS).3 Therefore, the majority of LS and OHP do not react productivelyand elute in fractions 2–6 and 27–30, respectively.Consistent with a cross-linked species, the intermediate fractionscontain both LS and HP (presumably with one sulfur atom inserted).This same cross-linked species is observed under turnover conditionswith unlabeled substrate (Figure S3), consistentwith sulfur insertion at C8 being rate-limiting.


Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase.

Lanz ND, Pandelia ME, Kakar ES, Lee KH, Krebs C, Booker SJ - Biochemistry (2014)

SDS-PAGE analysis offractions from DE-52 column. Lanes are molecularweight markers (M), flow-through during column load (A and B), andsamples from eluted 10 mL fractions as the NaCl gradient was increasedfrom 0.1–1 M (1–14). Fractions that were pooled andused in further experiments are boxed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216189&req=5

fig2: SDS-PAGE analysis offractions from DE-52 column. Lanes are molecularweight markers (M), flow-through during column load (A and B), andsamples from eluted 10 mL fractions as the NaCl gradient was increasedfrom 0.1–1 M (1–14). Fractions that were pooled andused in further experiments are boxed.
Mentions: To understand the natureof the turnover-dependent interaction between LS and its substrateand to provide evidence for a potential intermediate in catalysis,we studied the Ec LS reaction with [8,8,8-2H3]-OHP. Because sulfur insertion takes place at C6 beforeC8 and because quantitative deuterium substitution at C8 of the octanoylmoiety results essentially in arrest of turnover after the first sulfurinsertion, our working model would predict that OHP and LS shouldbecome cross-linked through the auxiliary cluster under turnover conditionswhen using [8,8,8-2H3]-OHP as substrate. Ec HP binds tightly to Ec LS; however,the two proteins can be separated by anion-exchange chromatography. Ec HP (pI = 4.59) adheres strongly to anion-exchange resinunder conditions described in Materials and Methods, whereas Ec LS (pI = 8.15) adheres poorly, if atall, under the same conditions. Figure 2A showsan SDS-PAGE analysis of fractions eluting from a Mono Q column underincreasing concentrations of NaCl. Ec LS and [8,8,8-2H3]-OHP were applied to the column after theircoincubation under turnover conditions in the absence of requiredlow-potential reductant. LS elutes essentially in the void volumeof the column (fractions 1–6), whereas [8,8,8-2H3]-OHP begins to elute at approximately 750 mM NaCl. The faintband that migrates slightly higher than [8,8,8-2H3]-OHP corresponds to LHP (or monothiolated LHP), which sometimescontaminates our preparations of apo-HP that are used to prepare OHP.When the proteins are incubated both in the presence of SAM and requiredreductant (sodium dithionite), a new species appears that displaysmigratory properties that are intermediate between those of LS andOHP (Figure 2B; fractions 20–25). Forreasons that we do not understand, OHP supports no more than 30–40%turnover (i.e., 0.3–0.4 equiv LHP per LS).3 Therefore, the majority of LS and OHP do not react productivelyand elute in fractions 2–6 and 27–30, respectively.Consistent with a cross-linked species, the intermediate fractionscontain both LS and HP (presumably with one sulfur atom inserted).This same cross-linked species is observed under turnover conditionswith unlabeled substrate (Figure S3), consistentwith sulfur insertion at C8 being rate-limiting.

Bottom Line: LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA(•)).Generation of the cross-linked species with a small, unlabeled (N(6)-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction.Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S](0) clusters.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and ‡Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.

ABSTRACT
Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N(6)-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-l-methionine (SAM) to l-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA(•)). In the LS reaction, two equivalents of 5'-dA(•) are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe-4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (N(6)-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe-4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe-4S](0) clusters.

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