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De Diego I.,European Molecular Biology Laboratory Hamburg | Kuper J.,European Molecular Biology Laboratory Hamburg | Kuper J.,Rudolf Virchow Center for Biomedical Research | Bakalova N.,European Molecular Biology Laboratory Hamburg | And 3 more authors.
Science Signaling | Year: 2010

Death-associated protein kinase (DAPK) provides a model for calcium-bound calmodulin (CaM)-dependent protein kinases (CaMKs). Here, we report the crystal structure of the binary DAPK-CaM complex, using a construct that includes the DAPK catalytic domain and adjacent autoregulatory domain. When DAPK was in a complex with CaM, the DAPK autoregulatory domain formed a long seven-turn helix. This DAPK-CaM module interacted with the DAPK catalytic domain through two separate domain-domain interfaces, which involved the upper and the lower lobe of the catalytic domain. When bound to DAPK, CaM adopted an extended conformation, which was different from that in CaM-CaMK peptide complexes. Complementary biochemical analysis showed that the ability of DAPK to bind CaM correlated with its catalytic activity. Because many features of CaM binding are conserved in other CaMKs, our findings likely provide a generally applicable model for regulation of CaMK activity. Copyright 2008 the American Association for the Advancement of Science; all rights reserved. Source

Burns K.E.,New York University | McAllister F.E.,Harvard University | Schwerdtfeger C.,Boston Biochem | Mintseris J.,Harvard University | And 8 more authors.
Journal of Biological Chemistry | Year: 2012

Dop is critical for the full virulence of Mycobacterium tuberculosis; however, its mechanism is not understood. Results: Asp-95 was identified as a catalytically significant residue. Conclusion: This work suggests that Asp-95 functions either as a direct nucleophile forming a unique anhydride intermediate or is part of a catalytic center that includes polarized water as the nucleophile. Significance: Understanding the mechanism of Dop can help guide the design and selection of inhibitors. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Elleuche S.,TU Hamburg - Harburg | Fodor K.,European Molecular Biology Laboratory Hamburg | Fodor K.,Eotvos Lorand University | Von Der Heyde A.,TU Hamburg - Harburg | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2014

NAD(P)+-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40°C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co2+. Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs. © 2013 Springer-Verlag. Source

Elleuche S.,TU Hamburg - Harburg | Fodor K.,European Molecular Biology Laboratory Hamburg | Fodor K.,Eotvos Lorand University | Klippel B.,TU Hamburg - Harburg | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2013

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni2+-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni2+, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD +/NADH, whereas no activity has been observed with NADP +/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn2+. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni2+-dependent activity towards 1,3-propanediol. © 2013 Springer-Verlag Berlin Heidelberg. Source

Gerner L.,University of Oslo | Munack S.,University of Oslo | Temmerman K.,European Molecular Biology Laboratory Hamburg | Temmerman K.,European Molecular Biology Laboratory Heidelberg | And 7 more authors.
Biochemical and Biophysical Research Communications | Year: 2016

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe267 is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe267Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in Escherichia coli. © 2016 Elsevier Inc. Source

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