Dataset Information

Crystal structures of the quinone oxidoreductase from Thermus thermophilus HB8 and its complex with NADPH: implication for NADPH and substrate recognition.

ABSTRACT: The crystal structures of the zeta-crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QOR(Tt)) and of its complex with NADPH have been determined at 2.3- and 2.8-A resolutions, respectively. QOR(Tt) is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOR(Ec)) and horse liver alcohol dehydrogenase. QOR(Tt) forms a homodimer in the crystal by interaction of the betaF-strands in domain II, forming a large beta-sheet that crosses the dimer interface. High thermostability of QOR(Tt) was evidenced by circular dichroic measurement. NADPH is located between the two domains in the QOR(Tt)-NADPH complex. The disordered segment involved in the coenzyme binding of apo-QOR(Tt) becomes ordered upon NADPH binding. The segment covers an NADPH-binding cleft and may serve as a lid. The 2'-phosphate group of the adenine of NADPH is surrounded by polar and positively charged residues in QOR(Tt), suggesting that QOR(Tt) binds NADPH more readily than NADH. The putative substrate-binding site of QOR(Tt), unlike that of QOR(Ec), is largely blocked by nearby residues, permitting access only to small substrates. This may explain why QOR(Tt) has weak p-benzoquinone reduction activity and is inactive with such large substrates of QOR(Ec) as 5-hydroxy-1,4-naphthoquinone and phenanthraquinone.

SUBMITTER: Shimomura Y

PROVIDER: S-EPMC164865 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Crystal structures of the quinone oxidoreductase from Thermus thermophilus HB8 and its complex with NADPH: implication for NADPH and substrate recognition.

Shimomura Yoshimitsu Y Kakuta Yoshimitsu Y Fukuyama Keiichi K

Journal of bacteriology 20030701 14

The crystal structures of the zeta-crystalline-like soluble quinone oxidoreductase from Thermus thermophilus HB8 (QOR(Tt)) and of its complex with NADPH have been determined at 2.3- and 2.8-A resolutions, respectively. QOR(Tt) is composed of two domains, and its overall fold is similar to the folds of Escherichia coli quinone oxidoreductase (QOR(Ec)) and horse liver alcohol dehydrogenase. QOR(Tt) forms a homodimer in the crystal by interaction of the betaF-strands in domain II, forming a large b ...[more]

PMID: 12837796

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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

Crystal structures of the quinone oxidoreductase from Thermus thermophilus HB8 and its complex with NADPH: implication for NADPH and substrate recognition.

Publications

Crystal structures of the quinone oxidoreductase from Thermus thermophilus HB8 and its complex with NADPH: implication for NADPH and substrate recognition.

Similar Datasets

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