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1.
The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi+). This process requires the formation of “supernucleophilic” Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.  相似文献   

2.
Yeast damage-associated response protein (Dap1p) and mouse progesterone receptor membrane component-1 protein (mPGRMC1p) belong to a highly conserved class of putative membrane-associated progesterone binding proteins (MAPR), with Dap1p and inner zone antigen (IZA), the rat homologue of mPGRMC1p, recently being reported to bind heme. While primary structure analysis reveals similarities to the cytochrome b(5) motif, neither of the two axial histidines responsible for ligation to the heme is present in any of the MAPR proteins. In this paper, EPR, MCD, CD, UV-vis, and general biochemical methods have been used to characterize the nature of heme binding in both Dap1p and a His-tagged, membrane anchor-truncated mPGRMC1p. As isolated, Dap1p is a tetramer which can be converted to a dimer upon addition of 150 mM salt. The heme is noncovalently attached, with a maximal, in vitro, heme loading of approximately 30%, for both proteins. CD and fluorescence spectroscopies indicate a well-ordered structure, suggesting the low level of heme loading is probably not due to improperly folded protein. EPR confirmed a five-coordinate, high-spin, ferric resting state for both proteins, indicating one axial amino acid ligand, in contrast to the six-coordinate, low-spin, ferric state of cytochrome b(5). The MCD spectrum confirmed this conclusion for Dap1p and indicated the axial ligand is most likely a tyrosine and not a histidine, or a cysteine; however, an aspartic acid residue could not be conclusively ruled out. Potential axial ligands, which are conserved in all MAPRs, were mutated (Y78F, D118A, and Y138F) and purified to homogeneity. The Y78F and D118A mutants were found to bind heme; however, Y138F did not. This result is consistent with the MCD data and indicates that Tyr138 is most likely the axial ligand to the heme in Dap1p.  相似文献   

3.
Successful iron acquisition plays a crucial role in bacterial virulence. Numerous Gram-negative pathogenic bacteria have developed a novel heme-acquisition system to steal iron from hosts. This system involves a cell-surface heme receptor, a periplasmic heme-transport protein (HTP) and inner-membrane proteins typical for ATP binding cassette transporters. We have cloned the gene encoding a periplasmic HTP from Pseudomonas aeruginosa, overexpressed it in Escherichia coli and purified it as a 33-kDa His-tagged protein. Heme-staining and heme-content assays reveal that the isolated HTP contains approximately 50% heme-bound and apo forms. The heme is noncovalently attached and can be transferred to apomyoglobin in vitro. Electron paramagnetic resonance and UV-vis spectroscopies indicate a five-coordinate, high-spin, ferric heme in HTP. HTP is reduced by dithionite but not by either dithiothreitol or ascorbate. Fluorescence and circular dichroism spectroscopies indicate a well-ordered structure for the HTP and a conformational change upon heme binding to apo-HTP. This was confirmed by limited proteolysis assays. Apo-HTP binds heme or protoporphyrin IX at 1:1 ratio with high affinity (K (d) approximately 1.2 and 14 nM, respectively). A BLASTP search revealed approximately 52 putative bacterial periplasmic heme transporters, which can be grouped into six classes, most of which are associated with pathogenic bacteria. Multiple sequence alignment reveals that these HTPs share low sequence similarity and no conserved common binding motif for heme ligation. However, a tyrosine residue (Y71) is highly conserved in the HTP sequences, which is likely an axial heme ligand in HTPs. Mutagenesis studies support Y71-heme iron ligation in the recombinant HTP.  相似文献   

4.
AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.  相似文献   

5.
Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.  相似文献   

6.
The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.  相似文献   

7.
Dispersin B (DspB), a family 20 beta-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves beta(1,6)-linked N-acetylglucosamine polymer. In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and nonconserved residues in the vicinity of the active site. The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsite -1. These residues were mutated using site-directed mutagenesis and characterized for their enzyme activity. Our results show that a highly conserved acid pair in beta-hexosaminidases D183 and E184, and E332 play a critical role in the hydrolysis of the substrates. pH activity profile analysis showed a shift to a higher pH (6.8) in the optimal activity for the E184Q mutant, suggesting that this residue might act as the acid/base catalyst. The reduction in k(cat) observed for Y187A and Y278A mutants suggests that the Y187 residue (unique to DspB) located on a loop might play a role in substrate specificity and be a part of subsite +1, whereas the hydrogen-bond interaction between Y278A and the N-acetyl group might help to stabilize the transition state. Mutation of W237 and W330 residues abolished hydrolytic activity completely suggesting that alteration at these positions might collapse the binding pocket for the N-acetyl group. Mutation of the conserved R27 residue (to R27A or R27K) also caused significant reduction in k(cat) suggesting that R27 might be involved in stabilization of the transition state. From these results, we conclude that in DspB, and possibly in other structurally similar family 20 hydrolases, some residues at the active site assist in orienting the N-acetyl group to participate in the substrate-assisted mechanism, whereas other residues such as R27 and E332 assist in holding the terminal N-acetylglucosamine during the hydrolysis.  相似文献   

8.
Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.  相似文献   

9.
We have analyzed the role of individual heme-ligating histidine residues for assembly of holo-cytochrome b6, and we show that the two hemes bL and bH bind in two subsequent steps to the apo-protein. Binding of the low-potential heme bL is a prerequisite for binding the high-potential heme bH. After substitution of His86, which serves as an axial ligand for heme bL, the apo-protein did not bind heme, while substitution of the heme bL-ligating residue His187 still allowed binding of both hemes. Similarly, after replacement of His202, one axial ligand to heme bH, binding of only heme bL was observed, whereas replacement of His100, the other heme bH ligand, resulted in binding of both hemes. These data indicate sequential heme binding during formation of the holo-cytochrome, and the two histidine residues, which serve as axial ligands to the same heme molecule (heme bL or heme bH), have different importance during heme binding and cytochrome assembly. Furthermore, determination of the heme midpoint potentials of the various cytochrome b6 variants indicates a cooperative adjustment of the heme midpoint potentials in cytochrome b6.  相似文献   

10.
11.
The determination of ligand binding properties is a key step in our understanding of gas sensing and discrimination by gas sensory proteins. HemAT is a newly discovered signal transducer heme protein that recognizes O(2) and discriminates against other gases such as CO and NO. We have used FTIR spectroscopy on CO- and NO-bound sensor domain HemAT and sensor domain distal mutants Y70F, T95A, R91A, and L92A to gain insight into the structure of the iron-bound ligand at ambient temperature. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand and also allow us to investigate the communication pathway between the distal residues of the protein and the heme. We show the formation of both H-bonded and non-H-bonded conformations in the CO-bound forms. In addition, we report the presence of multiple conformations in the NO-bound forms. Such distal H-bonding is crucial for ligand binding and activation by the heme. The comparison of the O(2), NO, and CO data demonstrates that Thr95 and Tyr70 are crucial for ligand recognition and discrimination and, thus, for specific sensing of gases, and L92 is crucial for controlling the conformational changes of the Thr95 and Tyr70 residues upon NO binding.  相似文献   

12.
Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.  相似文献   

13.
14.
To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.  相似文献   

15.
A detailed resonance Raman and electronic absorption investigation has been carried out on a series of novel distal and proximal variants of recombinant catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. In particular, variants of the distal triad Pro-Asp-Asn and the proximal triad His-Asp-Trp have been studied in their ferric and ferrous states at various pH. The data suggest marked differences in the structural role of the conserved residues and hydrogen-bond networks in KatG and CCP, which might be connected to the different catalytic activity. In particular, in KatG the proximal residues have a major role in the stability of the protein architecture because the disruption of the proximal Trp-Asp hydrogen bond by mutation weakens heme binding to the protein. On the distal side, replacing the hydrogen-acceptor carboxamide group of Asn153 by an aspartate carboxylate group or an aliphatic residue alters or disrupts the hydrogen bond with the distal His. As a consequence, the basicity of His123 is altered. The effect of mutation on Asp152 is noteworthy. Replacement of the Asp152 with Ser makes the architecture of the protein very similar to that of CCP. The Asp152 residue, which has been shown to be important in the hydrogen peroxide oxidation reaction, is expected to be hydrogen bonded to the nitrogen atom of Ile248 which is part of the KatG-specific insertion LL1, as in other KatGs. This insertion is at one edge of the heme, and connects the distal side with the proximal helices E and F, the latter carrying the proximal His ligand. We found that the distal Asp-Ile hydrogen bond is important for the stability of the heme architecture and its alteration changes markedly the proximal His-Asp hydrogen-bond interaction.  相似文献   

16.
The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

17.
Serpentine receptors with G-protein coupled receptor like seven transmembrane (7 TM) topology are identified in Plasmodium. A class of 7 TM receptors known as purinergic receptors binds to purines such as ADP, ATP and UTP and mediates important physiological functions including regulation of calcium signaling. Here we performed in silico analysis of Plasmodium falciparum serpentine receptors and found that one of the P. falciparum serpentine receptors, PfSR12 possess nucleotide binding consensus P-loop sequence in addition to seven transmembrane domains. The presence of conserved seven transmembrane domains and a consensus nucleotide binding sequence (P-loop) suggest that PfSR12 is a putative purinergic receptor. On further analysis using docking programmes we found four active binding residues Asn149, Lys150, Asn151 and Gly152 in P-loop of PfSR12, interact with ATP. This work gives insights into the interactions between putative purinergic receptor PfSR12 and its ligand ATP which can be explored in structure based drug designing against malaria.  相似文献   

18.
A relation between pH-induced conformational transitions of horse heart ferricytochrome c and the kinetics of external ligand coordination to heme iron was investigated by optical spectroscopy, circular dichroism and viscometry. The dependencies of both the association, k (a), and dissociation rate constants of cyanide binding on pH were determined from kinetic measurements. The association rate constant exhibits a bell-shaped form of dependence on pH in the region where this protein unfolds. The maximum of the dependence of k (a) on pH is found to be coincident with the pK values of conformational transitions of ferricytochrome c in solutions with both low and high ionic strengths. This observation is explained in terms of ferricytochrome c unfolding, which is characterized by two processes: the gradual opening of the heme crevice accompanied by the detachment of the axial Met80 and its replacement with a water molecule. The former process enhances the rate, whereas the latter results in the inhibition of the rate of cyanide binding.  相似文献   

19.
Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme catalyzing the hydrolysis of urea into ammonia and carbon dioxide. It is present in many bacteria, fungi, yeasts and plants. Most species, with few exceptions, use nickel metalloenzyme urease to hydrolyze urea, which is one of the commonly used nitrogen fertilizer in plant growth thus its enzymatic hydrolysis possesses vital importance in agricultural practices. Considering the essentiality and importance of urea and urease activity in most plants, this study aimed to comparatively investigate the ureases of two important legume species such as Glycine max (soybean) and Medicago truncatula (barrel medic) from Fabaceae family. With additional plant species, primary and secondary structures of 37 plant ureases were comparatively analyzed using various bioinformatics tools. A structure based phylogeny was constructed using predicted 3D models of G. max and M. truncatula, whose crystallographic structures are not available, along with three additional solved urease structures from Canavalia ensiformis (PDB: 4GY7), Bacillus pasteurii (PDB: 4UBP) and Klebsiella aerogenes (PDB: 1FWJ). In addition, urease structures of these species were docked with urea to analyze the binding affinities, interacting amino acids and atom distances in urease-urea complexes. Furthermore, mutable amino acids which could potentially affect the protein active site, stability and flexibility as well as overall protein stability were analyzed in urease structures of G. max and M. truncatula. Plant ureases demonstrated similar physico-chemical properties with 833–878 amino acid residues and 89.39–90.91 kDa molecular weight with mainly acidic (5.15–6.10 pI) nature. Four protein domain structures such as urease gamma, urease beta, urease alpha and amidohydro 1 characterized the plant ureases. Secondary structure of plant ureases also demonstrated conserved protein architecture, with predominantly α-helix and random coil structures. In structure-based phylogeny, plant ureases from G. max, M. truncatula and C. ensiformis were clearly diverged from bacterial ureases of B. pasteurii and K. aerogenes. Glu, Thr, His and Gly were commonly found as interacting residues in most urease-urea docking complexes while Glu was available in all docked structures. Besides, Ala and Arg residues, which are reported in active-site architecture of plant and bacterial ureases were present in G. max urea-urease complex but not present in others. Moreover, Arg435 and Arg437 in M. truncatula and G. max, respectively were identified as highly mutable hotspot residues residing in amidohydro 1 domain of enzyme. In addition, a number of stabilizing residues were predicted upon mutation of these hotspot residues however Cys and Thr made strong implications since they were also found in codon-aligned sequences as substitutions of hotspot residues. Comparative analyses of primary sequence and secondary structure in 37 different plants demonstrated quite conserved natures of ureases in plant kingdom. Structure-based phylogeny indicated the presence of a possible prokaryote-eukaryote split and implicated the subjection of bacterial ureases to heavy selection in prokaryotic evolution compared to plants. Urea-urease docking complexes suggested that different species could share common interacting residues as well as may have some other uncommon residues at species-dependent way. In silico mutation analyses identified mutable amino acids, which were predicted to reside in catalytic site of enzyme therefore mutagenesis at these sites seemed to have adverse effects on enzyme efficiency or function. This study findings will become valuable preliminary resource for future studies to further understand the primary, secondary and tertiary structures of urease sequences in plants as well as it will provide insights about various binding features of urea-urease complexes.  相似文献   

20.
The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 μM), with more hydrogen-bond contacts (ΔH = ?73.8 kJ/mol) and unfavourable entropy (?TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.  相似文献   

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