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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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Related in: MedlinePlus

Structure of the L. pneumophila dehydratase. Aribbon representation of the dimer is presented in panel a with themetal clusters depicted in space-filling representations. A stereoviewof one subunit of the enzyme is displayed in panel b. The N-terminalβ-domain is colored light blue, whereas the C-terminal αdomain is shown in purple. This figure and Figures 3 and 5–7 were prepared with PyMOL.33
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fig2: Structure of the L. pneumophila dehydratase. Aribbon representation of the dimer is presented in panel a with themetal clusters depicted in space-filling representations. A stereoviewof one subunit of the enzyme is displayed in panel b. The N-terminalβ-domain is colored light blue, whereas the C-terminal αdomain is shown in purple. This figure and Figures 3 and 5–7 were prepared with PyMOL.33

Mentions: Overall, the quality of the electrondensity for both polypeptidechains in the asymmetric unit was excellent with the exceptions ofseveral surface loops (breaks between Lys 161–Asn 167 and Ile250–Phe 259 in subunit A and between Asp 160–Asn 167and Lys 332–Ser 337 in subunit B). Shown in Figure 2a is a ribbon representation of the serine dehydratasedimer. It has overall dimensions of ∼100 Å × 80 Å× 60 Å and a total buried surface area of 4800 Å2. The iron–sulfur clusters are separated by ∼25Å. There are three cis-peptide bonds at Pro15, Pro 289, and Pro 395. Both Pro 15 and Pro 289 reside approximately10 Å from the active site, whereas Pro 395 abuts one side ofthe iron–sulfur cluster. Shown in Figure 2b is a stereoview of one subunit, which is distinctly bilobal inarchitecture. The N-terminal or β domain, delineated by Met1–Lys 161, is dominated by a five-stranded mixed β sheetthat is flanked on one side by four α helices. The C-terminaldomain, which harbors the active site cluster, is composed of 11 αhelices. Given that the α carbons for the two subunits of thedimer superimpose with a root-mean-square deviation of 0.25 Å,the following discussion will refer only to subunit B.


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)

Structure of the L. pneumophila dehydratase. Aribbon representation of the dimer is presented in panel a with themetal clusters depicted in space-filling representations. A stereoviewof one subunit of the enzyme is displayed in panel b. The N-terminalβ-domain is colored light blue, whereas the C-terminal αdomain is shown in purple. This figure and Figures 3 and 5–7 were prepared with PyMOL.33
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263429&req=5

fig2: Structure of the L. pneumophila dehydratase. Aribbon representation of the dimer is presented in panel a with themetal clusters depicted in space-filling representations. A stereoviewof one subunit of the enzyme is displayed in panel b. The N-terminalβ-domain is colored light blue, whereas the C-terminal αdomain is shown in purple. This figure and Figures 3 and 5–7 were prepared with PyMOL.33
Mentions: Overall, the quality of the electrondensity for both polypeptidechains in the asymmetric unit was excellent with the exceptions ofseveral surface loops (breaks between Lys 161–Asn 167 and Ile250–Phe 259 in subunit A and between Asp 160–Asn 167and Lys 332–Ser 337 in subunit B). Shown in Figure 2a is a ribbon representation of the serine dehydratasedimer. It has overall dimensions of ∼100 Å × 80 Å× 60 Å and a total buried surface area of 4800 Å2. The iron–sulfur clusters are separated by ∼25Å. There are three cis-peptide bonds at Pro15, Pro 289, and Pro 395. Both Pro 15 and Pro 289 reside approximately10 Å from the active site, whereas Pro 395 abuts one side ofthe iron–sulfur cluster. Shown in Figure 2b is a stereoview of one subunit, which is distinctly bilobal inarchitecture. The N-terminal or β domain, delineated by Met1–Lys 161, is dominated by a five-stranded mixed β sheetthat is flanked on one side by four α helices. The C-terminaldomain, which harbors the active site cluster, is composed of 11 αhelices. Given that the α carbons for the two subunits of thedimer superimpose with a root-mean-square deviation of 0.25 Å,the following discussion will refer only to subunit B.

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