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1.
Estevão A. Peroza Peter Güntert Eva Freisinger Oliver Zerbe 《Journal of molecular biology》2009,387(1):207-218
Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as ZnII, CuI, and CdII that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the βE-domain of the early cysteine-labeled protein (Ec-1) from wheat (βE-Ec-1), which represents the first three-dimensional structure of a plant MT. The βE-domain comprises the 51 C-terminal residues of Ec-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear ZnII binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three ZnII ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by 15N relaxation experiments support the results of the structure determination in that βE-Ec-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced ZnII with CdII ions and examined the effects of metal ion release on incubation with a metal ion chelator. 相似文献
2.
Eva Freisinger 《Journal of biological inorganic chemistry》2011,16(7):1035-1045
The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life
that have the ability to coordinate various transition metal ions, including ZnII, CdII, and CuI. The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four
subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between
the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to
assume that these differences will also be manifested on the structural level. This was already impressively demonstrated
with the elucidation of the three-dimensional structure of the wheat Ec-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so
far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus
is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination
of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long
linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid
of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as
the metal ion to cysteine ratios are important for predicting and understanding possible metal–thiolate cluster structures. 相似文献
3.
Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In
contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein
family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties
and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (Ec-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant Ec-1 is able to bind a total of six divalent d
10 metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable
to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset
of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic
digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence
of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing
11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal
ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily
so far.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
This article is dedicated to Prof. Bernhard Lippert on the occasion of his 60th birthday. 相似文献
4.
Montserrat Serra-Batiste Neus Cols Luis A. Alcaraz Antonio Donaire Pilar González-Duarte Milan Vašák 《Journal of biological inorganic chemistry》2010,15(5):759-776
Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2
(MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However,
its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of
recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands.
The fully metallated M8
II–MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn6–MTC and Cd7–MTC species from M8
II–MTC after treatment with Chelex 100. The NMR characterization of Cd7–MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal
or the four-metal thiolate cluster. Whereas the metal–Cys connectivities in the three-metal cluster located in the N-terminal
domain (residues 1–31) reveal a Cd3Cys9 cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32–64) precluded the determination
of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding
of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu8–9–MTC). The subsequent generation of Cu12–MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties
of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs. 相似文献
5.
Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a “cyclic reaction” was stimulated. Of metal ions tested they were effective in the order Pb2+ > Cu2+ > Zn2+ = Cd2+ > Ni2+ > Co2+. The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn2+-binding site(s). A mutant in which βHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn2+. Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn2+-induced FTIR difference spectra of the βHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that βHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase. 相似文献
6.
The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His244 or His266 residue. The binding of Zn2+ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co2+, Ca2+, Ni2+, Mg2+, and Mn2+. The strength of Ca2+, Co2+, Mg2+, Mn2+ and Ni2+ binding to the site close to His244 is stronger than that of these divalent metal ions binding to the site close to His266. 相似文献
7.
Rivka Goldshleger Guy Patchornik Meirav Bar Shimon Daniel M. Tal Robert L. Post Steven J. D. Karlish 《Journal of bioenergetics and biomembranes》2001,33(5):387-399
This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on / subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins. 相似文献
8.
Matthew Kennedy Lian Yu Maria João Lima Carla S. Ascenso Christopher Czaja Isabel Moura Jose J. G. Moura F. Rusnak 《Journal of biological inorganic chemistry》1998,3(6):643-649
Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)4 metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin
mutant proteins were reconstituted with Fe3+, Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process
that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides,
Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant
proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine
residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction
in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.
Received: 17 June 1998 / Accepted: 3 September 1998 相似文献
9.
Molecular dynamics (MD) simulations of the estrogen receptor DNA-binding domain (ERDBD) as a dimer in complex with its DNA
response element (ERE) show a significant difference in both structure and dynamics, compared to a MD simulation of monomeric
ERDBD bound to its half-site response element (EREH). The C-terminal zinc binding domain (ZnII), including a region (helix II) which is in a helical conformation in ERE-(ERDBD)2, is considerably more flexible in EREH-ERDBD than in the dimeric complex. In EREH-ERDBD, all helical hydrogen bonds in helix
II are broken and the entire ZnII region is detached from a hydrogen bonding network that in ERE-(ERDBD)2 connects to other parts of the protein as well as to the DNA. The regions that become flexible in EREH-ERDBD are identical
to the regions where the NMR solution structure of free ERDBD is poorly ordered. This strongly suggests that dimerisation
of ERDBD is required for ordering of the ZnII region and that monomeric binding to DNA is not sufficient for the ordering. This contrasts to the glucocorticoid receptor
DNA-binding domain (GRDBD) which has essentially the same mobility (uniform and limited), regardless of whether it is free
as a monomer in solution, bound as a monomer to its half-site response element or in a dimeric complex with the full response
element. The hydrogen bonding network that connects ZnII with other parts of the protein and to DNA is almost identical in ERDBD and GRDBD. However, in GRDBD there is also a serine
(in the N-terminal zinc coordinating region) with a central role in this network, connecting to the ZnII region. This serine is replaced by a glycine in ERDBD and we suggest that this substitution is sufficient for destabilisation
of the network, thus leading to a more flexible ZnII region, which becomes ordered first upon forming a complex with another ERDBD and DNA.
Received: 6 March 1998 / Revised version: 22 June 1998 / Accepted: 2 September 1998 相似文献
10.
Anikó Czene Eszter Németh István G. Zóka Noémi I. Jakab-Simon Tamás Körtvélyesi Kyosuke Nagata Hans E. M. Christensen Béla Gyurcsik 《Journal of biological inorganic chemistry》2013,18(3):309-321
Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX14NX8HX3H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446–576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn2+ ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn2+ ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485–487, 511–515 and 570–571, and in ΔN4-NColE7 those between residues 467–468, 530–535 and 570–571. 相似文献
11.
Duy Duc Nguyen Ramesh Pandian Doyoun Kim Sung Chul Ha Hye-Jin Yoon Kap Sun Kim Kyung Hee Yun Jin-Hahn Kim Kyeong Kyu Kim 《Biochemical and biophysical research communications》2014
The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co2+ rather than Zn2+: the kcat (s−1) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co2+, and Zn2+ conditions, respectively. Consistently, addition of low concentrations of Co2+ to PaAP previously saturated with Zn2+ greatly enhanced the enzymatic activity, suggesting that Co2+ may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co2+ or Zn2+ commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co2+- and Zn2+-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co2+ for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase. 相似文献
12.
Jianping Hu Ziheng Hu Yan Zhang Xiaojun Gou Ying Mu Lirong Wang Xiang-Qun Xie 《Journal of molecular modeling》2016,22(7):156
Nucleotide excision repair (NER) is a pivotal life process for repairing DNA nucleotide mismatch caused by chemicals, metal ions, radiation, and other factors. As the initiation step of NER, the xeroderma pigmentosum complementation group A protein (XPA) recognizes damaged DNA molecules, and recruits the replication protein A (RPA), another important player in the NER process. The stability of the Zn2+-chelated Zn-finger domain of XPA center core portion (i.e., XPA98-210) is the foundation of its biological functionality, while the displacement of the Zn2+ by toxic metal ions (such as Ni2+, a known human carcinogen and allergen) may impair the effectiveness of NER and hence elevate the chance of carcinogenesis. In this study, we first calculated the force field parameters for the bonded model in the metal center of the XPA98-210 system, showing that the calculated results, including charges, bonds, angles etc., are congruent with previously reported results measured by spectrometry experiments and quantum chemistry computation. Then, comparative molecular dynamics simulations using these parameters revealed the changes in the conformation and motion mode of XPA98-210 Zn-finger after the substitution of Zn2+ by Ni2+. The results showed that Ni2+ dramatically disrupted the relative positions of the four Cys residues in the Zn-finger structure, forcing them to collapse from a tetrahedron into an almost planar structure. Finally, we acquired the binding mode of XPA98-210 with its ligands RPA70N and DNA based on molecular docking and structural alignment. We found that XPA98-210’s Zn-finger domain primarily binds to a V-shaped cleft in RPA70N, while the cationic band in its C-terminal subdomain participates in the recognition of damaged DNA. In addition, this article sheds light on the multi-component interaction pattern among XPA, DNA, and other NER-related proteins (i.e., RPA70N, RPA70A, RPA70B, RPA70C, RPA32, and RPA14) based on previously reported structural biology information. Thus, we derived a putative cytotoxic mechanism associated with the nickel ion, where the Ni2+ disrupts the conformation of the XPA Zn-finger, directly weakening its interaction with RPA70N, and thus lowering the effectiveness of the NER process. In sum, this work not only provides a theoretical insight into the multi-protein interactions involved in the NER process and potential cytotoxic mechanism associated with Ni2+ binding in XPA, but may also facilitate rational anti-cancer drug design based on the NER mechanism. 相似文献
13.
The impact of Zn2+ ion on interactions of flavonols galangin (Gal), kaempferol (Kae), quercetin (Que) and myricetin (Myr) with bovine serum albumin (BSA) in aqueous solution were studied by fluorescence quenching technique. The results exhibited that Zn2+ ion affected significantly the interactions and the effect was distinct for the flavonol bearing different number of B-ring hydroxyl. Each flavonol can quench the fluorescence of BSA, displaying a quenching extent of Myr > Que > Kae > Gal, which is in good agreement with the number variation of the B-ring hydroxyl. The presence of Zn2+ ion promoted the quenching for the flavonols, exhibiting an extent of Que > Myr > Kae > Gal. The values of Ka for Kae, Que and Myr decreased whereas KSV and kq for Gal, Kae and Que increased with the number of B-ring hydroxyl. The type of BSA fluorescence quenching for Gal, Kae and Que hardly changed but the preference of static quenching increased. The values of KSV and kq for Myr remarkably decreased and the fluorescence quenching of BSA alternatively occurred via both static and dynamic type instead of only one (static or dynamic). The results suggest the key role of the B-ring hydroxyl and the distinct effect of its number in the interactions. Each flavonol may capture the BSA-bound ZnII in the solution, forming ZnII-flavonol complex that is possibly responsible for BSA fluorescence quenching. The B-ring hydroxyl could establish hydrogen bonds with BSA in the absence of Zn2+ and act as donors for chelating in the presence of Zn2+. The formation of dinuclear ZnII-Myr complex together with the hydrogen bonds between the free B-ring hydroxyl and BSA may contribute to the exceptional behavior of Myr. 相似文献
14.
Zhi-Hui Zhang 《Inorganica chimica acta》2007,360(8):2783-2788
The reactions of 5-(4-pyridyl)-1,3,4-oxadiazole-2-thiol (Hpyt) with typical octahedral metal ions (CoII and NiII) under different conditions generate a series of porous coordination polymers with the general formula of {[M(pyt)2(H2O)2] · (solvents)}n. In all these complexes, the versatile pyt anionic ligand behaves as the thioamide isomer with the μ-Npy,Noxa binding fashion, as confirmed by the X-ray crystallographic studies. Generally, the pyt linkers connect the metal centers to afford uniform 2-D grid-like host coordination frameworks, which stack in a parallel manner to engender the final 3-D crystalline lattices with 1-D open channels. Especially for CoII, different reactive routes and media lead to the productions of four analogous compounds with the inclusion of varied guest solvates, in one of which unusual water clusters are observed. 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2428-2436
Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2+. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger. 相似文献
16.
A new psychrophilic marine protease was found from a marine bacterium Flavobacterium YS-80 in the Chinese Yellow Sea. The protease is about 49 kD with an isoelectric point about 4.5. It consists of 480 amino acids and is homologous to a psychrophilic alkaline protease (PAP) from an Antarctic Pseudomonas species. The protein was purified from the natural bacterium fermented and crystallized. Its crystal structure (PDB ID 3U1R) was solved at 2.0 Å by Molecular Replacement using a model based on PAP, and was refined to a crystallographic Rwork of 0.16 and an Rfree of 0.21. The marine protease consists of a two domain structure with an N-terminal domain including residues 37–264 and a C-terminal domain including residues 265–480. Similar to PAP, the N-terminal domain is responsible for proteolysis and the C-terminal is for stability. His186, His190, His196 and Tyr226 are ligands for the Zn2+ ion in the catalytic center. The enzyme''s Tyr226 is closer to the Zn2+ ion than in PAP and it shows a stronger Zn2+―Tyr-OH bond. There are eight calcium ions in the marine protease molecule and they have significantly shorter bond distances to their ligands compared to their counterparts in all three crystal forms of PAP. On the other hand, the loops in the marine protease are more compact than in PAP. This makes the total structure stable and less flexible, resulting in higher thermo stability. These properties are consistent with the respective environments of the proteases. The structural analysis of this new marine protease provides new information for the study of psychrophilic proteases and is helpful for elucidating the structure-environment adaptation of these enzymes. 相似文献
17.
Claesson M Lindqvist Y Madrid S Sandalova T Fiskesund R Yu S Schneider G 《Journal of molecular biology》2012,417(4):279-293
The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn2+ and Mg2+ located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn2+-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn2+ in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn2+ acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155. 相似文献
18.
Zinc (Zn2+) was shown to invariably inhibit muscimol-stimulated36Cl− uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn2+ sensitivity of the GABAA receptor-gated36Cl− uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive
to Zn2+. Although diazepam-potentiation of muscimol-stimulated36Cl− uptake was unaltered by 100 μM Zn2+ in the cerebellum. Zn2+ inhibited [3H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni2+ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn2+ did not affect [3H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn2+ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [3H]Ro 15-4513 binding was not affected by Zn2+ or Ni2+ at any of the tested concentrations. These results suggest that the GABAA ligand-gated Cl− flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar
diazepam-insensitive [3H]Ro 15-4513 binding sites are insensitive to Zn2+ and Ni2+. 相似文献
19.
Thomas D. Pfister Amir Y. Mirarefi Alan J. Gengenbach Xuan Zhao Connor Danstrom Nicole Conatser Yi-Gui Gao Howard Robinson Charles F. Zukoski Andrew H.-J. Wang Yi Lu 《Journal of biological inorganic chemistry》2007,12(1):126-137
Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a MnII-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215–222, 1997; Gengenbach et al. in Biochemistry 38:11425–11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k
cat/K
M) than the variant in the original design, mostly due to a stronger K
M of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with CoII at the designed MnII site were also obtained. The metal ion in the engineered metal-binding site overlays well with MnII bound in MnP, suggesting that this variant is the closest structural model of the MnII-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the
metal; the metal–ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at
the designed site, difference in the identity of metal ions (CoII rather than MnII) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the
environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
20.
Xiao-Jun Zhao 《Inorganica chimica acta》2007,360(6):1970-1976
The coordination chemistry of a flexible poly(triazolyl)alkane derivative, fluconazole (HFlu), with a series of transition metal ions and dicyanamide (dca) anionic co-ligand has been explored to afford six new metal-organic coordination polymers. Complexes [M(HFlu)2(dca)2]n (M = MnII for 1, FeII for 2, CoII for 3, ZnII for 5, and CdII for 6) have the isostructural 1-D double-chain array via bridging fluconazole, whereas [Cu3(Flu)2(dca)4(CH3OH)2]n (4) shows an unusual 2-D layered metal-organic framework with dimeric CuII subunits. Notably, both types of coordination patterns are extended into distinct 3-D supramolecular networks via hydrogen-bonding interactions. This result indicates that the choice of metal ion has a significant effect on these polymeric structures as well as the binding modes of the ligands, which is discussed in detail. The ZnII and CdII complexes 5 and 6 display similar fluorescent emissions at 260 nm in the solid state, which essentially are intraligand transitions. 相似文献