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 Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Cytosolic Phospholipase A2α–deficient Mice Are Resistant to Collagen-induced Arthritis

2003 ◽  
Vol 197 (10) ◽  
pp. 1297-1302 ◽  
Author(s):  
Martin Hegen ◽  
Linhong Sun ◽  
Naonori Uozumi ◽  
Kazuhiko Kume ◽  
Mary E. Goad ◽  
...  
Keyword(s):  
Critical Role ◽  
Wild Type ◽  
Collagen Induced Arthritis ◽  
Cytosolic Phospholipase ◽  
Severity Scores ◽  
In The Wild ◽  
Cytosolic Phospholipase A2α ◽  
Antibody Levels ◽  
Deficient Mice

Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2α (cPLA2α) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2α plays a key role in the development of CIA, we backcrossed cPLA2α-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2α-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2α-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2α-deficient mice. Although the disease scores differed significantly between cPLA2α mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2α in the pathogenesis of CIA.

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.

The development of the sensory neuron pattern in the antennal disc of wild-type and mutant (lz3, ssa) Drosophila melanogaster

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 1063-1075
Author(s):  
M.C. Lienhard ◽  
R.F. Stocker
Keyword(s):  
Drosophila Melanogaster ◽  
Sensory Neuron ◽  
Antennal Segment ◽  
Wild Type ◽  
Homeotic Mutant ◽  
In The Wild ◽  
The Brain ◽  
Antennal Disc ◽  
Antennal Nerve

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents.

Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains

Development ◽  
2002 ◽  
Vol 129 (6) ◽  
pp. 1327-1338 ◽  
Author(s):  
Masanori Takahashi ◽  
Noriko Osumi
Keyword(s):  
Expression Patterns ◽  
Domain Formation ◽  
Wild Type ◽  
Whole Embryo Culture ◽  
Neuronal Markers ◽  
Signalling Molecules ◽  
Neuronal Progenitors ◽  
In The Wild ◽  
Mutant Rat

Recent studies have shown that generation of different kinds of neurones is controlled by combinatorial actions of homeodomain (HD) proteins expressed in the neuronal progenitors. Pax6 is a HD protein that has previously been shown to be involved in the differentiation of the hindbrain somatic (SM) motoneurones and V1 interneurones in the hindbrain and/or spinal cord. To investigate in greater depth the role of Pax6 in generation of the ventral neurones, we first examined the expression patterns of HD protein genes and subtype-specific neuronal markers in the hindbrain of the Pax6 homozygous mutant rat. We found that Islet2 (SM neurone marker) and En1 (V1 interneurone marker) were transiently expressed in a small number of cells, indicating that Pax6 is not directly required for specification of these neurones. We also observed that domains of all other HD protein genes (Nkx2.2, Nkx6.1, Irx3, Dbx2 and Dbx1) were shifted and their boundaries became blurred. Thus, Pax6 is required for establishment of the progenitor domains of the ventral neurones. Next, we performed Pax6 overexpression experiments by electroporating rat embryos in whole embryo culture. Pax6 overexpression in the wild type decreased expression of Nkx2.2, but ectopically increased expression of Irx3, Dbx1 and Dbx2. Moreover, electroporation of Pax6 into the Pax6 mutant hindbrain rescued the development of Islet2-positive and En1-positive neurones. To know reasons for perturbed progenitor domain formation in Pax6 mutant, we examined expression patterns of Shh signalling molecules and states of cell death and cell proliferation. Shh was similarly expressed in the floor plate of the mutant hindbrain, while the expressions of Ptc1, Gli1 and Gli2 were altered only in the progenitor domains for the motoneurones. The position and number of TUNEL-positive cells were unchanged in the Pax6 mutant. Although the proportion of cells that were BrdU-positive slightly increased in the mutant, there was no relationship with specific progenitor domains. Taken together, we conclude that Pax6 regulates specification of the ventral neurone subtypes by establishing the correct progenitor domains.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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