Dataset Information

Structure of D-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme-ligand interactions.

ABSTRACT: D-Alanine-D-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two D-Ala molecules using ATP to produce D-Ala-D-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 A resolution), TtDdl-adenylyl imidodiphosphate (2.6 A), TtDdl-ADP (2.2 A), TtDdl-ADP-D-Ala (1.9 A) and TtDdl-ATP-D-Ala-D-Ala (2.3 A). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdl-complex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism.

SUBMITTER: Kitamura Y

PROVIDER: S-EPMC2756165 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Structure of D-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme-ligand interactions.

Kitamura Yoshiaki Y Ebihara Akio A Agari Yoshihiro Y Shinkai Akeo A Hirotsu Ken K Kuramitsu Seiki S

Acta crystallographica. Section D, Biological crystallography 20090916 Pt 10

D-Alanine-D-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two D-Ala molecules using ATP to produce D-Ala-D-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 A resolution), TtDdl-adenylyl imidodiphosphate (2.6 A), TtDdl-ADP (2.2 A), TtDdl-ADP-D-Ala ...[more]

PMID: 19770507

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Project description:Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To further characterize functional significance, we successfully mapped 113 acetylation sites on their 54 authentic and 59 homologous protein structures. The acetylation in the majority of proteins occurs in ordered structures and the sites were situated near the negatively charged glutamic acid residues. In addition, 59 of 103 acetylations were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylations. Finally, we further summarized 22 critical acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and protein-protein interaction. The structural information of 113 acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins. Data processing, bioinformatics: MS and MS/MS spectral data were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The carbamidomethylation of cysteine was selected as a fixed modification. The oxidation of methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified.

Dataset Information

Structure of D-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme-ligand interactions.

Publications

Structure of D-alanine-D-alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme-ligand interactions.

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