Side‐chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L‐leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4–6% of capacity) while remaining remarkably responsive to ADP/L‐leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L‐leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain (‘antenna’), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg‐1 change essentially replicated the Arg443Ser‐1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443‐1 or Arg409‐1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser‐1 mutant protein, obtained in recombinant form, regained most of the wild‐type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg‐2 mutant acquired most of the hGDH1 properties. Hence, side‐chain interactions between 409 and 443 positions in the ‘antenna’ region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.

     

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.     

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors— erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine—was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme–inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors—derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.     

Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution

Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (DeltaT(1/2) 相似文献   

Molecular insight into substrate recognition by human cytosolic sialidase NEU2

Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide α(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide α(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid.     

The diversity of FtsY‐lipid interactions

The bacterial signal recognition particle (SRP) receptor FtsY forms a complex with the SRP Ffh to target nascent polypeptide chains to the bacterial inner membrane. How FtsY interacts with lipids and associates to the membrane is unclear. Here, we show that vesicle binding leads to partial protection against proteolytic degradation and a change in secondary structure, which differs depending on whether the lipids are simple mixtures of zwitterionic and anionic lipids, mimics of Escherichia coli lipids, or lysolipids. Lipid binding alters the stability of FtsY. Thermal unfolding of FtsY in buffer shows two transitions, one occurring at ～60°C and the other at ～90°C. The thermal intermediate accumulating between 60 and 90°C has structural features in common with the state induced by binding to E. coli lipids. E. coli lipid extract induces a single transition around 70°C, anionic lipids have no effect while cooperative unfolding is completely removed in lysolipids. Thus, the lipid environment profoundly influences the dynamic properties of FtsY, leading to three different kinds of FtsY‐lipid interactions with different effects on structure, proteolytic protection, and stability, and is driven both by hydrophobic and electrostatic interactions. Trypsin digestion experiments highlight the central role of the N‐domain in lipid contacts, whereas the A‐ and G‐domains appear to play a more minor part. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 595–606, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Role of C-terminal region of Staphylococcal nuclease for foldability,stability, and activity

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.     

Expression,purification and characterisation of a novel mutant of the human protein kinase CK2

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant subunit. According to our theoretical predictions, we observed that the -helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.     

Experimental and theoretical studies of the three-dimensional structure of human interleukin-4

The structure of human interleukin 4 (IL-4) was predicted utilizing a series of experimental and theoretical techniques. Circular Dichroism (CD) spectroscopy indicated that IL-4 belonged to the all alpha-helix class of protein structures. Secondary structure prediction, site-directed mutagenesis, and CD spectroscopy suggested a predominantly alpha-helical structure, consistent with a four-helix bundle structural motif. A human/mouse IL-4 chimera was constructed to qualitatively evaluate alternative secondary structure predictions. The four predicted helices were assembled into tertiary structures using established algorithms. The mapping of three disulfide bridges in IL-4 provided additional constraints on possible tertiary structures. Using accessible surface contact area as a criterion, the most suitable structures were right handed all antiparallel four-helix bundles with two overhand loop connections. Successful loop closure and incorporation of the three disulfide constraints were possible while maintaining the expected shape, solvent accessibility, and steric interactions between loops and helices. Lastly, energy minimization was used to regularize the chain.     

Docking of human interleukin-15 to its specific receptor alpha chain: correlation between molecular modeling and mutagenesis experimental data

A structural model of the sushi domain of IL-15Ralpha was first obtained by homology modeling to study its interactions with IL-15 by means of molecular modeling, peptide scanning, and site-directed mutagenesis. From these experimental data, a putative interacting surface of IL-15Ralpha with a previously published IL-15 model was inferred: Leu25, Leu44, and Glu46 of IL-15 and Arg35 of IL-15Ralpha were found to be key interfacial residues and were subsequently used as filters for the construction of docking solutions. Human IL-15/IL-15Ralpha complexes were constructed in two stages, with a preliminary docking procedure, treating the two partners as rigid bodies and using these filters. In this first stage, two classes of docking solutions were characterized. From a topological point of view, each solution could be derived from the other by reverse orientation of one partner in relation to the other. In a second stage, several further energy refinements clearly favored one solution. Moreover, this unique docking solution was confirmed by molecular modeling of IL-15 mutants previously built and tested in our laboratory. Finally, this complex model, which is a useful tool to study the IL-15/IL-15Ralpha interface, was topologically compared to IL-2/IL-2Ralpha complexes (previous model in the literature and recent crystal structure).     

丁酰胆碱酯酶结构研究新进展

丁酰胆碱酯酶(butyrylcholinesterase, BChE, EC 3.1.1.8),能与有机磷毒剂或杀虫剂结合,并能水解许多酯类、肽类及酰胺类化合物,对这些化合物的中毒具有防治作用.近年来通过计算机模拟技术及定点突变技术对其结构研究取得了重要进展,对人BChE外周阴离子部位的结构有了新的认识, 并通过氨基酸替换使BChE获得了水解有机磷酸酐的新功能.     

Identification of the active site residues in ATP‐citrate lyase's carboxy‐terminal portion

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.     

Circular dichroism spectroscopic study of non‐covalent interactions of poly‐L‐glutamic acid with a porphyrin derivative in aqueous solutions

The interactions of poly‐L ‐glutamic acid and a cationic porphyrin derivative in aqueous solutions were studied by the combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies. It was found that non‐covalent interactions between both agents influence the structure of the polymeric matrix and the guest porphyrins and vice versa, but the physico‐chemical properties of the solutions, especially the pH and the relative permittivity of the solvent, play a key role in the structure of the polypeptide part of the formed complexes. It was shown that the interaction with porphyrins prevents the precipitation of poly‐L ‐glutamic acid in aqueous solution at acidic pH. In special conditions, the porphyrins attached to the polypeptide probably possess face‐to‐face interaction as demonstrated by the enhancement of the characteristic ECD signal and the appearance of sidebands on its short and long wavelength sides. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of amylose content by structure‐based modification of OsGBSS1 activity in rice (Oryza sativa L.)

The rice Waxy (Wx) gene encodes granule‐bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site‐directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch‐binding capacity, its ADP‐glucose‐binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.     

Concerted action of activation-induced cytidine deaminase and uracil-DNA glycosylase reduces covalently closed circular DNA of duck hepatitis B virus

Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision.