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Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Thoden JB, Holden HM, Grant GA - Biochemistry (2014)

Bottom Line: A number of highly conserved or invariant residues found in the β domain are clustered around the iron-sulfur center.His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster.Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.

ABSTRACT
Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S](2+) cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal β domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron-sulfur cluster producing a "tail in mouth" configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the β domain are clustered around the iron-sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

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Inhibitionkinetics of the L. pneumophila dehydratasewith l-cysteine (top) and d-serine (bottom). Forthe left panels, the l-serine concentration was varied atdifferent fixed concentrations of inhibitor and double-reciprocalplots of velocity vs l-serine concentration are shown: (topleft) 0 (●), 50 (○), 100 (◆), 150 (▲),200 (▼), and 300 (filled half-square) μM l-cysteineand (bottom left) 0 (●), 5 (○), 25 (◆), 50 (▲),100 (▼), and 200 (filled half-square) mM d-serine.The middle panels shows slope replots of data from the double-reciprocalplots. The right panels show plots of velocity vs inhibitor concentrationat fixed substrate concentrations: (top right) varying l-cysteineconcentrations with 25 (●), 50 (○), and 100 (◆)mM l-serine and (bottom right) varying d-serineconcentrations at 2 (●), 10 (○), and 50 (◆) mM l-serine. The y-axes of the double-reciprocalplots are reciprocal velocity with the velocity measured as the changein absorbance at 250 nm per minute. Double-reciprocal plots were fitby linear regression analysis that yielded values for the slopes.Slope replots and plots of velocity vs inhibitor concentration ata constant substrate concentration were fit either by linear regressionanalysis or in the case of d-serine to a second-degree polynomial.The values for Ki were estimated graphicallyfrom the x-axis intercept, which equals −Ki. The data points in plots of velocity vs inhibitorconcentration were connected from point to point using the smoothfit function of Kaleidograph.
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fig4: Inhibitionkinetics of the L. pneumophila dehydratasewith l-cysteine (top) and d-serine (bottom). Forthe left panels, the l-serine concentration was varied atdifferent fixed concentrations of inhibitor and double-reciprocalplots of velocity vs l-serine concentration are shown: (topleft) 0 (●), 50 (○), 100 (◆), 150 (▲),200 (▼), and 300 (filled half-square) μM l-cysteineand (bottom left) 0 (●), 5 (○), 25 (◆), 50 (▲),100 (▼), and 200 (filled half-square) mM d-serine.The middle panels shows slope replots of data from the double-reciprocalplots. The right panels show plots of velocity vs inhibitor concentrationat fixed substrate concentrations: (top right) varying l-cysteineconcentrations with 25 (●), 50 (○), and 100 (◆)mM l-serine and (bottom right) varying d-serineconcentrations at 2 (●), 10 (○), and 50 (◆) mM l-serine. The y-axes of the double-reciprocalplots are reciprocal velocity with the velocity measured as the changein absorbance at 250 nm per minute. Double-reciprocal plots were fitby linear regression analysis that yielded values for the slopes.Slope replots and plots of velocity vs inhibitor concentration ata constant substrate concentration were fit either by linear regressionanalysis or in the case of d-serine to a second-degree polynomial.The values for Ki were estimated graphicallyfrom the x-axis intercept, which equals −Ki. The data points in plots of velocity vs inhibitorconcentration were connected from point to point using the smoothfit function of Kaleidograph.

Mentions: The amino acids that display significant inhibition ofenzymatic activity are l-cysteine, d-serine, l-histidine, and glycine. Strikingly, l-alanine wasnot an effective inhibitor. Double-reciprocal plots of activity varying l-serine at fixed inhibitor concentrations are most consistentwith simple competitive inhibition for l-cysteine and l-histidine with Ki values of 60μM and 11.4 mM, respectively (Figure 4). The slope replots are linear as are plots of velocity versus inhibitorconcentration, indicating that the velocity goes to zero at an infiniteinhibitor concentration, which is consistent with simple competitiveinhibition (Scheme 2, dashed box). Interestingly, l-cystine does not inhibit the enzyme at 1 mM and only showsapproximately 12% inhibition at 20 mM.


Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Thoden JB, Holden HM, Grant GA - Biochemistry (2014)

Inhibitionkinetics of the L. pneumophila dehydratasewith l-cysteine (top) and d-serine (bottom). Forthe left panels, the l-serine concentration was varied atdifferent fixed concentrations of inhibitor and double-reciprocalplots of velocity vs l-serine concentration are shown: (topleft) 0 (●), 50 (○), 100 (◆), 150 (▲),200 (▼), and 300 (filled half-square) μM l-cysteineand (bottom left) 0 (●), 5 (○), 25 (◆), 50 (▲),100 (▼), and 200 (filled half-square) mM d-serine.The middle panels shows slope replots of data from the double-reciprocalplots. The right panels show plots of velocity vs inhibitor concentrationat fixed substrate concentrations: (top right) varying l-cysteineconcentrations with 25 (●), 50 (○), and 100 (◆)mM l-serine and (bottom right) varying d-serineconcentrations at 2 (●), 10 (○), and 50 (◆) mM l-serine. The y-axes of the double-reciprocalplots are reciprocal velocity with the velocity measured as the changein absorbance at 250 nm per minute. Double-reciprocal plots were fitby linear regression analysis that yielded values for the slopes.Slope replots and plots of velocity vs inhibitor concentration ata constant substrate concentration were fit either by linear regressionanalysis or in the case of d-serine to a second-degree polynomial.The values for Ki were estimated graphicallyfrom the x-axis intercept, which equals −Ki. The data points in plots of velocity vs inhibitorconcentration were connected from point to point using the smoothfit function of Kaleidograph.
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Related In: Results  -  Collection

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fig4: Inhibitionkinetics of the L. pneumophila dehydratasewith l-cysteine (top) and d-serine (bottom). Forthe left panels, the l-serine concentration was varied atdifferent fixed concentrations of inhibitor and double-reciprocalplots of velocity vs l-serine concentration are shown: (topleft) 0 (●), 50 (○), 100 (◆), 150 (▲),200 (▼), and 300 (filled half-square) μM l-cysteineand (bottom left) 0 (●), 5 (○), 25 (◆), 50 (▲),100 (▼), and 200 (filled half-square) mM d-serine.The middle panels shows slope replots of data from the double-reciprocalplots. The right panels show plots of velocity vs inhibitor concentrationat fixed substrate concentrations: (top right) varying l-cysteineconcentrations with 25 (●), 50 (○), and 100 (◆)mM l-serine and (bottom right) varying d-serineconcentrations at 2 (●), 10 (○), and 50 (◆) mM l-serine. The y-axes of the double-reciprocalplots are reciprocal velocity with the velocity measured as the changein absorbance at 250 nm per minute. Double-reciprocal plots were fitby linear regression analysis that yielded values for the slopes.Slope replots and plots of velocity vs inhibitor concentration ata constant substrate concentration were fit either by linear regressionanalysis or in the case of d-serine to a second-degree polynomial.The values for Ki were estimated graphicallyfrom the x-axis intercept, which equals −Ki. The data points in plots of velocity vs inhibitorconcentration were connected from point to point using the smoothfit function of Kaleidograph.
Mentions: The amino acids that display significant inhibition ofenzymatic activity are l-cysteine, d-serine, l-histidine, and glycine. Strikingly, l-alanine wasnot an effective inhibitor. Double-reciprocal plots of activity varying l-serine at fixed inhibitor concentrations are most consistentwith simple competitive inhibition for l-cysteine and l-histidine with Ki values of 60μM and 11.4 mM, respectively (Figure 4). The slope replots are linear as are plots of velocity versus inhibitorconcentration, indicating that the velocity goes to zero at an infiniteinhibitor concentration, which is consistent with simple competitiveinhibition (Scheme 2, dashed box). Interestingly, l-cystine does not inhibit the enzyme at 1 mM and only showsapproximately 12% inhibition at 20 mM.

Bottom Line: A number of highly conserved or invariant residues found in the β domain are clustered around the iron-sulfur center.His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster.Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.

ABSTRACT
Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a β domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S](2+) cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal β domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron-sulfur cluster producing a "tail in mouth" configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the β domain are clustered around the iron-sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Show MeSH
Related in: MedlinePlus