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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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Possible location the allosteric binding pocket of L. pneumophila dehydratase. The catalytic domain is highlightedin orange, whereasthe β domain is displayed in green.
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fig6: Possible location the allosteric binding pocket of L. pneumophila dehydratase. The catalytic domain is highlightedin orange, whereasthe β domain is displayed in green.

Mentions: Clearly,Cys 458 serves as an excellent mimic for the locationof the substrate, l-serine. As indicated in Scheme 1, dehydration of l-serine is assisted byextraction of the proton from its α carbon by a catalytic base.Given that there are no potential catalytic bases close enough tothe α carbon of Cys 458 to perform this function in the structuredescribed here, our current model represents a “precatalytic”conformation. There are two histidine residues (His 19 and His 61)within approximately 6 Å of the Cys 458 α carbon, however,that could possibly move within catalytic distance upon a conformationalrearrangement initiated by serine binding (Figure 6). Mutation of His 61 to alanine results in a complete lossof catalytic activity. Importantly, the Fe–S cluster is stillintact because the charge transfer absorbance (∼400 nm) isnot diminished (not shown), thus suggesting that His 61 may serveas the catalytic base. Mutation of His 19 to alanine has an effecton both Km and kcat. In addition, the mutation has a significant effect onthe Ki for l-cysteine and essentiallyno effect on the Ki for d-serine.Most likely, His 19 is involved in substrate binding because the mutationmainly affects the interaction with the competitive inhibitor, l-cysteine.


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)

Possible location the allosteric binding pocket of L. pneumophila dehydratase. The catalytic domain is highlightedin orange, whereasthe β domain is displayed in green.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Possible location the allosteric binding pocket of L. pneumophila dehydratase. The catalytic domain is highlightedin orange, whereasthe β domain is displayed in green.
Mentions: Clearly,Cys 458 serves as an excellent mimic for the locationof the substrate, l-serine. As indicated in Scheme 1, dehydration of l-serine is assisted byextraction of the proton from its α carbon by a catalytic base.Given that there are no potential catalytic bases close enough tothe α carbon of Cys 458 to perform this function in the structuredescribed here, our current model represents a “precatalytic”conformation. There are two histidine residues (His 19 and His 61)within approximately 6 Å of the Cys 458 α carbon, however,that could possibly move within catalytic distance upon a conformationalrearrangement initiated by serine binding (Figure 6). Mutation of His 61 to alanine results in a complete lossof catalytic activity. Importantly, the Fe–S cluster is stillintact because the charge transfer absorbance (∼400 nm) isnot diminished (not shown), thus suggesting that His 61 may serveas the catalytic base. Mutation of His 19 to alanine has an effecton both Km and kcat. In addition, the mutation has a significant effect onthe Ki for l-cysteine and essentiallyno effect on the Ki for d-serine.Most likely, His 19 is involved in substrate binding because the mutationmainly affects the interaction with the competitive inhibitor, l-cysteine.

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