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排序方式: 共有197条查询结果,搜索用时 156 毫秒
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124.
Mirza O Skov LK Sprogøe D van den Broek LA Beldman G Kastrup JS Gajhede M 《The Journal of biological chemistry》2006,281(46):35576-35584
The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a beta-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336-344 and 132-137) in the proximity of the active site move up to 16 and 4 A, respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops. 相似文献
125.
Pedersen L Hansen K Nielsen J Lantz AE Thykaer J 《Biotechnology and bioengineering》2012,109(1):116-124
The market for glucoamylase is large and very competitive and the production process has been optimized through several decades. So far a thorough characterization of the process has not been published, but previous academic reports suggest that the process suffers from severe byproduct formation. In this study we have carried out a thorough characterization of a process as close as possible to the industrial reality. The results show that the oxygen-limited phases of the process have the highest glucoamylase yields on carbon and that the byproducts are efficiently reused in late phases of the process. An alternative process with low glucose concentration show that high osmolarity is beneficial for the process, and we conclude that oxygen limitation, high osmolarity, and the associated byproduct metabolism are important for the efficiency of the process. 相似文献
126.
Krintel C Frydenvang K Olsen L Kristensen MT de Barrios O Naur P Francotte P Pirotte B Gajhede M Kastrup JS 《The Biochemical journal》2012,441(1):173-178
Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6?μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1?kcal≈4.187?kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46?mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders. 相似文献
127.
Helle Hald Philip K. Ahring Tommy Liljefors Jette S. Kastrup 《Journal of molecular biology》2009,391(5):906-178
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory neurotransmission. Upon glutamate application, 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid receptors undergo rapid and almost complete desensitization that can be attenuated by positive allosteric modulators. The molecular mechanism of positive allosteric modulation has been elucidated previously by crystal structures of the ligand-binding core of iGluR2 in complex with, for example, cyclothiazide (CTZ). Here, we investigate the structure and function of CTZ and three closely related analogues NS1493, NS5206, and NS5217 at iGluR2, by X-ray crystallography and fast application patch-clamp electrophysiology. CTZ was the most efficacious and potent modulator of the four compounds on iGluR2(Q)i [Emax normalized to response of glutamate: 754% (CTZ), 490% (NS1493), 399% (NS5206), and 476% (NS5217) and EC50 in micromolar: 10 (CTZ), 26 (NS1493), 43 (NS5206), and 48 (NS5217)]. The four modulators divide into three groups according to efficacy and desensitization kinetics: (1) CTZ increases the peak current efficacy twice as much as the three analogues and nearly completely blocks receptor desensitization; (2) NS5206 and NS5217 have low efficacy and only attenuate desensitization partially; (3) NS1493 has low efficacy but nearly completely blocks receptor desensitization. A hydrophobic substituent at the 3-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system is important for compound efficacy, with the following ranking: norbornenyl (bicyclo[2.2.1]hept-2-ene) > cyclopentyl > methyl. The replacement of the norbornenyl moiety with a significantly less hydrophobic cyclopentane ring increases the flexibility of the modulator as the cyclopentane ring adopts various conformations at the iGluR2 allosteric binding site. The main structural feature responsible for a nearly complete block of desensitization is the presence of an NH hydrogen bond donor in the 4-position of the 1,1-dioxo-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine ring system, forming an anchoring hydrogen bond to Ser754. Therefore, the atom at the 4-position of CTZ seems to be a major determinant of receptor desensitization kinetics. 相似文献
128.
Karla Frydenvang L. Leanne Lash Peter Naur Pekka A. Postila Darryl S. Pickering Caleb M. Smith Michael Gajhede Makoto Sasaki Ryuichi Sakai Olli T. Pentika?nen Geoffrey T. Swanson Jette S. Kastrup 《The Journal of biological chemistry》2009,284(21):14219-14229
The prevailing structural model for ligand activation of ionotropic
glutamate receptors posits that agonist efficacy arises from the stability and
magnitude of induced domain closure in the ligand-binding core structure. Here
we describe an exception to the correlation between ligand efficacy and domain
closure. A weakly efficacious partial agonist of very low potency for
homomeric iGluR5 kainate receptors, 8,9-dideoxyneodysiherbaine (MSVIII-19),
induced a fully closed iGluR5 ligand-binding core. The degree of relative
domain closure, ∼30°, was similar to that we resolved with the
structurally related high affinity agonist dysiherbaine and to that of
l-glutamate. The pharmacological activity of MSVIII-19 was
confirmed in patch clamp recordings from transfected HEK293 cells, where
MSVIII-19 predominantly inhibits iGluR5-2a, with little activation apparent at
a high concentration (1 mm) of MSVIII-19 (<1% of mean
glutamate-evoked currents). To determine the efficacy of the ligand
quantitatively, we constructed concentration-response relationships for
MSVIII-19 following potentiation of steady-state currents with concanavalin A
(EC50 = 3.6 μm) and on the nondesensitizing receptor
mutant iGluR5-2b(Y506C/L768C) (EC50 = 8.1 μm).
MSVIII-19 exhibited a maximum of 16% of full agonist efficacy, as measured in
parallel recordings with glutamate. Molecular dynamics simulations and
electrophysiological recordings confirm that the specificity of MSVIII-19 for
iGluR5 is partly attributable to interdomain hydrogen bond residues
Glu441 and Ser721 in the iGluR5-S1S2 structure. The
weaker interactions of MSVIII-19 with iGluR5 compared with dysiherbaine,
together with altered stability of the interdomain interaction, may be
responsible for the apparent uncoupling of domain closure and channel opening
in this kainate receptor subunit.Ionotropic glutamate receptors
(iGluRs)3 are central
to fast excitatory synaptic transmission in the central nervous system and are
involved in numerous physiological and pathophysiological processes. The
iGluRs consist of three different classes of receptors,
N-methyl-d-aspartic acid (NMDA),
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate
receptors (1), which are
assembled as tetramers in a dimer of dimers configuration
(2,
3). These receptors can be
considered as multidomain proteins, composed of an extracellular
N-terminal domain, a ligand-binding core made of discontinuous S1 and
S2 segments that form two lobes (domains D1 and D2), three
transmembrane-spanning regions (M1–M3) with a re-entrant loop between M1
and M2, and finally a cytoplasmic region
(1).Ligand-binding cores of iGluRs assume tertiary structures in solution that
reproduce the pharmacological profiles of full-length receptors.
Crystallographic studies of ligand-binding core complexes from representative
members of all three iGluR subtypes
(4–6)
as well as the ligand-binding core of the structurally related δ2
subunit in complex with d-serine
(7) have yielded unprecedented
insight into structural correlates of iGluR function. Binding of agonists to
iGluR ligand-binding cores can be described as a “Venus flytrap”
mechanism. In the resting state, the ligand-binding core is present in an open
form that is stabilized by antagonists
(4,
8,
9). When an agonist binds to
the ligand-binding core, a rotational change in conformation occurs, resulting
in domain closure of the D1 and D2 lobes around a central hinge region
(4,
6). In full-length receptors,
this domain closure is thought to result in the opening of the ion channel
(receptor activation). The extents of domain closure of ligand-binding cores
of AMPA and kainate receptor subunits are correlated with the activation and
the desensitization of the receptor
(9,
10).However, previous studies have questioned the association between the
degree of domain closure of the ligand-binding core and channel opening or
agonist efficacy. For example, AMPA was shown to induce a more closed
structure of the ligand-binding core of the mutated iGluR2(L650T) than was
expected from its partial agonist efficacy
(11,
12). Also, no correlation
between domain closure and agonist efficacy has been demonstrated for the NR1
subunit of NMDA receptors
(13).In this study, we present the first example of a nonmutated kainate
receptor that lacks the correlation between domain closure and efficacy. We
tested if two structurally related kainate receptor ligands, one an agonist
and one described previously as an antagonist
(14), conformed to the
prevailing structural model of ligand-induced activity. The high affinity
agonist dysiherbaine (DH) is a natural excitotoxin originally isolated from a
marine sponge (15,
16), whereas
8,9-dideoxyneodysiherbaine (MSVIII-19) is a synthetic analog that inhibits
activation of iGluR5 receptors
(14). To investigate receptor
interactions with the two closely related compounds as well as the degree of
domain closure introduced by the compounds, we determined the crystal
structures of DH and MSVIII-19 in complex with the ligand-binding core of the
kainate receptor subunit iGluR5 (iGluR5-S1S2). These two structures, along
with functional studies, provide novel insights into the mechanism of kainate
receptor activation, inhibition, and desensitization. 相似文献
129.
Jette Thykaer Jens Nielsen Wolfgang Wohlleben Tilmann Weber Michael Gutknecht Anna E. Lantz Evi Stegmann 《Metabolic engineering》2010,12(5):455-461
Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed. 相似文献
130.
Giuseppe Gallo Rosa Alduina Giovanni Renzone Jette Thykaer Linda Bianco Anna Eliasson-Lantz Andrea Scaloni Anna Maria Puglia 《Microbial cell factories》2010,9(1):95