scholarly journals Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

2005 ◽  
Vol 71 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Zhi-Wei Chen ◽  
Cheng-Ying Jiang ◽  
Qunxin She ◽  
Shuang-Jiang Liu ◽  
Pei-Jin Zhou
Keyword(s):  
Cytoplasmic Membrane ◽  
Sulfur Oxidation ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Wild Type ◽  
Immune Electron Microscopy ◽  
Mutant Proteins ◽  
Close Proximity ◽  
Catalytic Sites

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

scholarly journals A Zinc Ion Controls Assembly and Stability of the Major Capsid Protein of Rotavirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3595-3601 ◽  
Author(s):  
Inge Erk ◽  
Jean-Claude Huet ◽  
Mariela Duarte ◽  
Stéphane Duquerroy ◽  
Felix Rey ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Chelating Agent ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
High Concentration ◽  
Mutant Proteins

ABSTRACT The recent determination of the crystal structure of VP6, the major capsid protein of rotavirus, revealed a trimer containing a central zinc ion coordinated by histidine 153 from each of the three subunits. The role of the zinc ion in the functions of VP6 was investigated by site-directed mutagenesis. The mutation of histidine 153 into a serine (H153S and H153S/S339H) did not prevent the formation of VP6 trimers. At pH <7.0, about the pK of histidine, wild-type and mutated VP6 proteins display similar properties, giving rise to identical tubular and spherical assemblies. However, at pH >7.0, histidine 153 mutant proteins did not assemble into the characteristic 45-nm-diameter tubes, in contrast to wild-type VP6. These observations showed that under conditions in which histidine residues are not charged, the properties of VP6 depended on the presence of the centrally coordinated zinc atom in the trimer. Indeed, wild-type VP6 depleted of the zinc ion by a high concentration (100 mM) of a metal-chelating agent behaved like the H153 mutant proteins. The susceptibility of wild-type VP6 to proteases is greatly increased in the absence of zinc. NH2-terminal sequencing of the proteolytic fragments showed that they all contained the β-sheet-rich VP6 head domain, which appeared to be less sensitive to protease activity than the α-helical basal domain. Finally, the mutant proteins assembled well on cores, as demonstrated by both electron microscopy and rescue of transcriptase activity. Zinc is thus not necessary for the transcription activity. All of these observations suggest that, in solution, VP6 trimers present a structural flexibility that is controlled by the presence of a zinc ion.

scholarly journals Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM

2003 ◽  
Vol 372 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Teijo PELLINEN ◽  
Helena AHLFORS ◽  
Nicolas BLOT ◽  
Guy CONDEMINE
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Transmembrane Segments

The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure. A model of this protein, based on sequence similarity and the amphipathy profile, was constructed. The model depicts a β-barrel composed of 14 antiparallel β-strands. The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis. The protein has seven surface-exposed loops. They are rather small with the exception of one, loop L6. Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM. Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins. With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

Role of Cysteine Residues in the N-Terminal Extracellular Domain of the Human VIP 1 Receptor for Ligand Binding A Site-directed Mutagenesis Studya

2006 ◽  
Vol 805 (1) ◽  
pp. 585-589 ◽  
Author(s):  
PASCALE GAUDIN ◽  
ALAIN COUVINEAU ◽  
JEAN-JOSÉ MAORET ◽  
CHRISTIANE ROUYER-FESSARD ◽  
MARC LABURTHE
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
A Site

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes

1990 ◽  
Vol 10 (12) ◽  
pp. 6257-6263
Author(s):  
A Frankel ◽  
P Welsh ◽  
J Richardson ◽  
J D Robertus
Keyword(s):  
Enzyme Activity ◽  
Glutamic Acid ◽  
Structural Information ◽  
Wild Type ◽  
Toxin A ◽  
Mutant Proteins ◽  
Ricin Toxin ◽  
Enzymatic Inactivation ◽  
A Chain

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.

scholarly journals A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

1997 ◽  
Vol 326 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
Regina M. DAY ◽  
Ekaterina V. TORCHILIN ◽  
William W. BACHOVCHIN ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Chemical Shifts ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Imidazolium Cation ◽  
Nmr Study ◽  
In The Wild ◽  
Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Albino mutants of Streptomyces glaucescens tyrosinase

1991 ◽  
Vol 274 (3) ◽  
pp. 707-713 ◽  
Author(s):  
M P Jackman ◽  
A Hajnal ◽  
K Lerch
Keyword(s):  
Active Site ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Carbonyl Derivatives ◽  
Hydrogen Bonding Interactions ◽  
Albino Phenotype ◽  
Derivatives Of ◽  
Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

Structural Requirements of Human Ether-a-go-go–  related Gene Channels for Block by Bupivacaine

2007 ◽  
Vol 106 (3) ◽  
pp. 523-531 ◽  
Author(s):  
Cornelia C. Siebrands ◽  
Patrick Friederich
Keyword(s):  
Aromatic Amino Acids ◽  
Selectivity Filter ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Related Gene ◽  
Wild Type ◽  
Aromatic Residues ◽  
Ic50 Value ◽  
Herg Channels

Background Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. Methods Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. Results Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P &lt; 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P &lt; 0.001). Conclusions The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

scholarly journals Interaction of PomB with the Third Transmembrane Segment of PomA in the Na+-Driven Polar Flagellum of Vibrio alginolyticus

2004 ◽  
Vol 186 (16) ◽  
pp. 5281-5291 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Shingo Maki ◽  
Michio Homma
Keyword(s):  
Marine Bacterium ◽  
Vibrio Alginolyticus ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
The Third ◽  
Close Proximity ◽  
Flagellar Rotation ◽  
Lines Of Evidence

ABSTRACT The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na+-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na+ than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na+ conduction to flagellar rotation.

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