Category Archives: J Biol Chem

Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca.

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Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca.

J Biol Chem. 2017 Apr 14;:

Authors: Kumar H, Nguyen QT, Binda C, Mattevi A, Fraaije MW

Abstract
F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasingly attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420:NADPH oxidoreductase (Tfu-FNO) from T. fusca, being the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide-binding domain and a smaller C-terminal do-main. Residues interacting with the 2′-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr28, Ser50, Arg51, and Arg55 are important for discriminating between NADP+ and NAD+. Interestingly, a T28A mutant increased the kinetic efficiency more than three-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.

PMID: 28411200 [PubMed – as supplied by publisher]

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Characterization of the functional roles of amino acid residues in acceptor binding subsite +1 in the active site of the glucansucrase GTF180 enzyme of Lactobacillus reuteri 180.

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Characterization of the functional roles of amino acid residues in acceptor binding subsite +1 in the active site of the glucansucrase GTF180 enzyme of Lactobacillus reuteri…

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Procollagen Lysyl Hydroxylase 2 Expression is Regulated by an Alternative Downstream Transforming Growth Factor Beta-1 Activation Mechanism.

Procollagen Lysyl Hydroxylase 2 Expression is Regulated by an Alternative Downstream Transforming Growth Factor Beta-1 Activation Mechanism.

J Biol Chem. 2015 Oct 2;

Authors: Gjaltema RA, de Rond S, Rots MG, Bank RA

Abstract
PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) is a transforming growth factor beta-1 (TGFβ1) responsive gene that hydroxylates lysyl residues in collagen telopeptides and is essential for collagen pyridinoline cross-link formation in fibrotic pathologies. In this report we examined the molecular processes underlying TGFβ1-induced PLOD2 expression. We found that binding of the TGFβ1 pathway related transcription factors SMAD3 and SP1 mediated TGFβ1 enhanced PLOD2 expression and could be correlated to an increase of acetylated histone H3 and H4 at the PLOD2 promoter. Interestingly, the classical co-activators of SMAD3 complexes, p300 and CBP, were not responsible for the enhanced H3 and H4 acetylation. Depletion of SMAD3 reduced PLOD2 acetylated H3 and H4, indicating that another as of yet unidentified histone acetyltransferase binds to SMAD3 at PLOD2. Assessing histone methylation marks at the PLOD2 promoter depicted an increase of the active histone mark H3K79me2, a decrease of the repressive H4K20me3 mark, but no role for the generally strong transcription-related modifications: H3K4me3, H3K9me3 and H3K27me3. Collectively, our findings reveal that TGFβ1 induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter other than the currently known TGFβ1 downstream co-activators and epigenetic modifications. This also suggests that additional activation strategies are used downstream of the TGFβ1 pathway, and hence their unraveling could be of great importance to fully understand TGFβ1 activation of genes.

PMID: 26432637 [PubMed – as supplied by publisher]

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Role of the cytosolic loop C2 and the C-terminus of YidC in ribosome binding and insertion activity.

Role of the cytosolic loop C2 and the C-terminus of YidC in ribosome binding and insertion activity.
J Biol Chem. 2015 May 28;
Authors: Geng Y, Kedrov A, Caumanns JJ, Crevenna AH, Lamb DC, Beckmann R, Driessen AJ
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Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh.

Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh.

J Biol Chem. 2015 Apr 28;

Authors: Hänelt I, Jensen S, Wunnicke D, Slotboom DJ

Abstract
GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters (EAATs) that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions, and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apo-protein (Kd 120 mM), with a particularly low kon value (5 M-1s-1). At least two Na+ ions bind prior to aspartate. The binding of Na+ requires very high activation energy (Ea 106.8 kJmol-1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range and the aspartate binding was fast (kon of 1.4*105 M-1s-1), with low Ea and Q10 values (42.6 KJmol-1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding.

PMID: 25922069 [PubMed – as supplied by publisher]

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Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes.

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes.
J Biol Chem. 2015 Apr 20;
Authors: Tietz S, Puthiyaveetil S, Enlow HM, Yarbrough R, Wood M, Semchonok DA, Lowry T, Li Z, Jahns … Continue reading

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The bipartite Rac1 guanine nucleotide exchange factor engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) protects endothelial cells from apoptosis in blood vessel development.

The bipartite Rac1 guanine nucleotide exchange factor engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) protects endothelial cells from apoptosis in blood vessel…

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The solution structure, binding properties, and dynamics of the bacterial siderophore-binding protein FepB.

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The solution structure, binding properties, and dynamics of the bacterial siderophore-binding protein FepB.
J Biol Chem. 2014 Oct 17;289(42):29219-34
Authors: Chu BC, Otten R, Krewulak KD, Mulder FA,… Continue reading

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A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining β-glucosidases.

A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining β-glucosidases.

J Biol Chem. 2014 Oct 24;

Authors: Kallemeijn WW, Witte MD, Voorn-Brouwer TM, Walvoort MT, Li KY, Codée JD, van der Marel GA, Boot RG, Overkleeft HS, Aerts JM

Abstract
Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach, putative catalytic residues are firstly substituted to noncarboxylic amino-acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

PMID: 25344605 [PubMed – as supplied by publisher]

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Residue L940 has a crucial role in the linkage and reaction specificity of the glucansucrase GTF180 of the probiotic bacterium Lactobacillus reuteri 180.

Residue L940 has a crucial role in the linkage and reaction specificity of the glucansucrase GTF180 of the probiotic bacterium Lactobacillus reuteri 180.

J Biol Chem. 2014 Oct 6;

Authors: Meng X, Dobruchowska JM, Pijning T, Lόpez CA, Kamerling JP, Dijkhuizen L

Abstract
Highly conserved GH70 family glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue L940 in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in L940 of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physico-chemical properties [containing 67% to 100% of (α1→6) linkages] were produced by GTF180 and its L940 mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue L940 in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.

PMID: 25288798 [PubMed – as supplied by publisher]

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Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation.

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Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation.

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Crystal Structure of the Ectoine Hydroxylase: a Snapshot of the Active Site.

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Crystal Structure of the Ectoine Hydroxylase: a Snapshot of the Active Site.

J Biol Chem. 2014 Aug 29;

Authors: Höeppner A, Widderich N, Lenders M, Bremer E, Smits SH

Abstract
Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by Bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homo-dimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-hydroxyectoine on the other hand, is coordinated by EctD in manner different from that found in high-affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.

PMID: 25172507 [PubMed – as supplied by publisher]

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Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA.

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Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA.

J Biol…

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Control of the Diadenylate Cyclase CdaS in Bacillus subtilis: An Autoinhibitory Domain Limits c-di-AMP Production.

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Control of the Diadenylate Cyclase CdaS in Bacillus subtilis: An Autoinhibitory Domain Limits c-di-AMP Production.

J Biol Chem. 2014 Jun 16;

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Crystal structure of α-1,4-glucan lyase, a unique glycoside hydrolase family member with a novel catalytic mechanism.

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Crystal structure of α-1,4-glucan lyase, a unique glycoside hydrolase family member with a novel catalytic mechanism.

J Biol Chem. 2013 Sep 13;288(37):26764-74

Authors: Rozeboom HJ, Yu S, Madrid S, Kalk KH, Zhang R, Dijkstra BW

Abstract
α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)8-barrel catalytic domain with B and B’ subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

PMID: 23902768 [PubMed – indexed for MEDLINE]

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Physicochemical factors controlling the activity and energy coupling of an ionic strength-gated ATP-binding cassette (ABC) transporter.

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Physicochemical factors controlling the activity and energy coupling of an ionic strength-gated ATP-binding cassette (ABC) transporter.
J Biol Chem. 2013 Oct 11;288(41):29862-71
Authors: Karasawa A, Swier LJ, … Continue reading

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Decreased affinity of rhTRAIL-D269H/E195R to OPG overcomes TRAIL-resistance mediated by the bone microenvironment.

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Decreased affinity of rhTRAIL-D269H/E195R to OPG overcomes TRAIL-resistance mediated by the bone microenvironment.
J Biol Chem. 2013 Nov 26;
Authors: Bosman MC, Reis CR, Schuringa JJ, Vellenga E, Quax WJ
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Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.

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Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.

J Biol Chem….

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The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron-sulfur cluster protein assembly.

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The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron-sulfur cluster protein assembly.
J Biol Chem. 2012 Dec 21;287(52):43351-8
Authors: van Wietmarschen N, Moradian A, Morin GB, Lansd… Continue reading

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Structure and mode of peptide binding of pheromone receptor PrgZ.

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Structure and mode of peptide binding of pheromone receptor PrgZ.

J Biol Chem. 2012 Oct 26;287(44):37165-70

Authors: Berntsson RP, Schuurman-Wolters GK,…

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