Publications by authors named "Antje Keim"

8 Publications

  • Page 1 of 1

Characterization of the Domain Orientations of E. coli 5'-Nucleotidase by Fitting an Ensemble of Conformers to DEER Distance Distributions.

Structure 2016 Jan 24;24(1):43-56. Epub 2015 Dec 24.

Center for Structural Biology, Vanderbilt University, Nashville, TN 37212, USA; Department of Chemistry, Vanderbilt University, Nashville, TN 37212, USA; Vanderbilt University, 7330 Stevenson Center, Station B 351822, Nashville, TN 37235, USA. Electronic address:

Escherichia coli 5'-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate).
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http://dx.doi.org/10.1016/j.str.2015.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706771PMC
January 2016

Crystal structure of hexokinase KlHxk1 of Kluyveromyces lactis: a molecular basis for understanding the control of yeast hexokinase functions via covalent modification and oligomerization.

J Biol Chem 2010 Dec 12;285(52):41019-33. Epub 2010 Oct 12.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, University of Leipzig, D-04103 Leipzig, Germany.

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases.
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http://dx.doi.org/10.1074/jbc.M110.185850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003401PMC
December 2010

Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases.

FASEB J 2011 Jan 10;25(1):89-98. Epub 2010 Sep 10.

Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

Eukaryotic ATP-dependent 6-phosphofructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ∼ 1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full-length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (αβγ)(4) dodecamer of D(2) symmetry with dimensions of 161 × 157 × 233 Å mainly via interactions of the α chains. The N-terminal domains of the α and β chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2-fold axis form a (αβ)(2) dimer as does the core Pfk domains; however, the domains are swapped across the tetramerization interface. In PpPfk, the unique γ subunit participates in oligomerization of the αβ chains. This modulator protein was acquired from an ancient S-adenosylmethionine-dependent methyltransferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfks.
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http://dx.doi.org/10.1096/fj.10-163865DOI Listing
January 2011

Structure and function of the abasic site specificity pocket of an AP endonuclease from Archaeoglobus fulgidus.

DNA Repair (Amst) 2009 Feb 5;8(2):219-31. Epub 2008 Dec 5.

Institute of Biochemistry, Faculty of Biology, Pharmacy and Psychology, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

The major AP endonuclease in Escherichia coli Exonuclease III (ExoIII) is frequently used in gene technology due to its strong exonucleolytic activity. A thermostabilized variant of ExoIII or a homologous enzyme from thermophilic organisms could be most useful for further applications. For this purpose we characterized a nuclease from the hyperthermophilic archaeon Archaeoglobus fulgidus (Af_Exo), which shares 33% overall sequence identity and 55% similarity to ExoIII. The gene coding for this thermostable enzyme was cloned and expressed in E. coli. The purified protein shows a strong Mg(2+)-dependent nicking activity at AP-sites, nicking of undamaged double-stranded (ds) DNA and a weak exonucleolytic activity. A V217G variant of the enzyme was crystallized with decamer ds-DNA molecule, and the three-dimensional structure was determined to 1.7A resolution. Besides our goal to find or produce a thermostable exonuclease, the structural and catalytic data of Af_Exo and a series of mutant proteins, based on the crystal structure, provide new insight into the mechanism of abasic site recognition and repair. Each of the hydrophobic residues Phe 200, Trp 215 and Val 217, forming a binding pocket for the abasic deoxyribose in Af_Exo, were mutated to glycine or serine. By expanding the size of the binding pocket the unspecific endonucleolytic activity is increased. Thus, size and flexibility of the mostly hydrophobic binding pocket have a significant influence on AP-site specificity. We suggest that its tight fitting to the flipped-out deoxyribose allows for a preferred competent binding of abasic sites. In a larger or more flexible pocket however, intact nucleotides more easily bind in a catalytically competent conformation, resulting in loss of specificity. Moreover, with mutations of Phe 200 and Trp 215 we induced a strong exonucleolytic activity on undamaged DNA.
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http://dx.doi.org/10.1016/j.dnarep.2008.10.009DOI Listing
February 2009

Active-site mobility revealed by the crystal structure of arylmalonate decarboxylase from Bordetella bronchiseptica.

J Mol Biol 2008 Mar 5;377(2):386-94. Epub 2008 Jan 5.

Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

Arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica catalyzes the enantioselective decarboxylation of arylmethylmalonates without the need for an organic cofactor or metal ion. The decarboxylation reaction is of interest for the synthesis of fine chemicals. As basis for an analysis of the catalytic mechanism of AMDase and for a rational enzyme design, we determined the X-ray structure of the enzyme up to 1.9 A resolution. Like the distantly related aspartate or glutamate racemases, AMDase has an aspartate transcarbamoylase fold consisting of two alpha/beta domains related by a pseudo dyad. However, the domain orientation of AMDase differs by about 30 degrees from that of the glutamate racemases, and also significant differences in active-site structures are observed. In the crystals, four independent subunits showing different conformations of active-site loops are present. This finding is likely to reflect the active-site mobility necessary for catalytic activity.
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http://dx.doi.org/10.1016/j.jmb.2007.12.069DOI Listing
March 2008

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida.

FEBS J 2007 Nov;274(21):5767-79

Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.
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http://dx.doi.org/10.1111/j.1742-4658.2007.06102.xDOI Listing
November 2007

Crystallization and preliminary X-ray diffraction studies of hexokinase KlHxk1 from Kluyveromyces lactis.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 May 20;63(Pt 5):430-3. Epub 2007 Apr 20.

Biotechnologisch-Biomedizinisches Zentrum, Institut für Bioanalytische Chemie, Fakultät für Chemie und Mineralogie, Universität Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany.

Glucose acts as both a carbon source and a hormone-like regulator of gene expression in eukaryotic organisms from yeast to man. Phosphorylation of glucose is executed by hexokinases, which represent a class of multifunctional enzymes that, in addition to their contribution to the uptake and initiation of metabolism of glucose, fructose and mannose, are involved in glucose signalling. The genome of the budding yeast Kluyveromyces lactis encodes a single hexokinase (KlHxk1) and a single glucokinase (KlGlk1). KlHxk1 exists in a monomer-homodimer equilibrium which is presumed to play a role in metabolic regulation. In order to evaluate the physiological significance of KlHxk1 dimerization on a molecular level, the enzyme was crystallized and subjected to X-ray structure analysis. Crystallization employing ammonium sulfate, diammonium phosphate or polyethylene glycol 6000 at pH values of 8.0-9.5 gave seven different crystal forms of KlHxk1. Crystallographic data to 1.66 A resolution were obtained using synchrotron radiation. Structure determination of KlHxk1 in various packing environments will reveal the full architecture of the homodimeric enzyme and complete our mechanistic understanding of the catalytic and regulatory functions of the enzyme.
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http://dx.doi.org/10.1107/S1744309107018271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335009PMC
May 2007

Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Dec 30;62(Pt 12):1290-3. Epub 2006 Nov 30.

Center for Biotechnology and Biomedicine, Institute for Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.

Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 A using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes.
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http://dx.doi.org/10.1107/S1744309106050548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2225370PMC
December 2006