Dataset Information

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

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 ? domain and a ? 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 ? domain and is situated between this domain and the N-terminal ? 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 ? 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.

SUBMITTER: Thoden JB

PROVIDER: S-EPMC4263429 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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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 James B JB Holden Hazel M HM Grant Gregory A GA

Biochemistry 20141124 48

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 ...[more]

PMID: 25380533

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Project description:The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

Dataset Information

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

Publications

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

Similar Datasets

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