scholarly journals Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

2021 ◽  
Author(s):  
Yuanyuan Xia ◽  
Xiaoyu Li ◽  
Linli Yang ◽  
Xiaozhou Luo ◽  
Wei Shen ◽  
...  
Keyword(s):  
Rational Design ◽  
Function Analysis ◽  
Single Point ◽  
Single Point Mutation ◽  
Wild Type ◽  
Product Concentration ◽  
Sucrose Phosphorylase ◽  
Dynamic Simulations ◽  
Key Points ◽  
High Level

Abstract Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure–function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. Key points • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants

scholarly journals Cryo-EM structure of the human TACAN channel in a closed state

2021 ◽  
Author(s):  
xiaozhe chen ◽  
Yaojie Wang ◽  
Yang Li ◽  
Xuhang Lu ◽  
jianan chen ◽  
...  
Keyword(s):  
Single Point ◽  
Molecular Assembly ◽  
Activation Mechanism ◽  
Single Point Mutation ◽  
Globular Domain ◽  
Wild Type ◽  
Dynamic Simulations ◽  
Closed State ◽  
Conduction Pathway ◽  
Evolutionarily Conserved

TACAN is an ion channel involved in sensing mechanical pain. It has recently been shown to represent a novel and evolutionarily conserved class of mechanosensitive channels. Here, we present the cryoelectron microscopic structure of human TACAN (hTACAN). hTACAN forms a dimer in which each protomer consists of a transmembrane globular domain (TMD) that is formed of six helices and an intracellular domain (ICD) that is formed of two helices. Molecular dynamic simulations suggest that a putative ion conduction pathway is located inside each protomer. Single point mutation of the key residue Met207 significantly increased the surface tension activated currents. Moreover, cholesterols were identified at the flank of each subunit. Our data show the molecular assembly of hTACAN and suggest that the wild type hTACAN is in a closed state, providing a basis for further understanding the activation mechanism of the hTACAN channel.

scholarly journals Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bingran Wang ◽  
Tiancheng Lou ◽  
Lingling Wei ◽  
Wenchan Chen ◽  
Longbing Huang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Single Point ◽  
Sodium Dodecyl ◽  
Cross Resistance ◽  
Molecular Characteristics ◽  
Single Point Mutation ◽  
Wide Range ◽  
Significant Difference ◽  
Leaf Blights ◽  
High Level

AbstractAlternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

scholarly journals An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum

2021 ◽  
Vol 15 ◽  
Author(s):  
Esther Suk King Lai ◽  
Hisako Nakayama ◽  
Taisuke Miyazaki ◽  
Takanobu Nakazawa ◽  
Katsuhiko Tabuchi ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Synapse Formation ◽  
Single Point ◽  
Autism Spectrum ◽  
Mutant Mice ◽  
Single Point Mutation ◽  
Synapse Elimination ◽  
Wild Type ◽  
Post Mortem Brain ◽  
And Function

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10–15 (P10–15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

scholarly journals Definitive demonstration by synthesis of genome annotation completeness

2019 ◽  
Vol 116 (48) ◽  
pp. 24206-24213 ◽  
Author(s):  
Paul R. Jaschke ◽  
Gabrielle A. Dotson ◽  
Kay S. Hung ◽  
Diane Liu ◽  
Drew Endy
Keyword(s):  
Essential Gene ◽  
Single Point ◽  
Viable Cell ◽  
Essential Genes ◽  
Single Point Mutation ◽  
Evolutionary Adaptation ◽  
Wild Type ◽  
Bioinformatic Tools ◽  
Design And Synthesis ◽  
New Genes

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available—23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1727-1727
Author(s):  
Vanja Karamatic Crew ◽  
Carole Green ◽  
Stephen Parsons ◽  
Belinda K. Singleton ◽  
Geoff Daniels ◽  
...  
Keyword(s):  
Cell Line ◽  
Dna Analysis ◽  
Single Point ◽  
Jurkat Cells ◽  
Erythroid Cell ◽  
Single Point Mutation ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Hemopoietic Cells ◽  
Elevated Expression

Abstract Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS2002;99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

scholarly journals High-Level Chromosomally Mediated Tetracycline Resistance in Neisseria gonorrhoeae Results from a Point Mutation in the rpsJ Gene Encoding Ribosomal Protein S10 in Combination with the mtrR and penB Resistance Determinants

2005 ◽  
Vol 49 (10) ◽  
pp. 4327-4334 ◽  
Author(s):  
Mei Hu ◽  
Sobhan Nandi ◽  
Christopher Davies ◽  
Robert A. Nicholas
Keyword(s):  
Amino Acids ◽  
Neisseria Gonorrhoeae ◽  
Ribosomal Protein ◽  
Point Mutation ◽  
Single Point ◽  
Ribosomal Subunit ◽  
Tetracycline Resistance ◽  
Saturation Mutagenesis ◽  
Single Point Mutation ◽  
High Level

ABSTRACT Neisseria gonorrhoeae becomes resistant to tetracycline by two major mechanisms: expression of a plasmid-encoded TetM protein and mutations in endogenous genes (chromosomally mediated resistance). Early studies by Sparling and colleagues (P. F. Sparling F. A. J. Sarubbi, and E. Blackman, J. Bacteriol. 124:740-749, 1975) demonstrated that three genes were involved in high-level chromosomally mediated tetracycline resistance (MIC of tetracycline ≥ 2 μg/ml): ery-2 (now referred to as mtrR), penB, and tet-2. While the identities of the first two genes are known, the tet-2 gene has not been identified. We cloned the tet-2 gene, which confers tetracycline resistance, from tetracycline-resistant clinical isolate N. gonorrhoeae FA6140 and show that resistance is due to a single point mutation (Val-57 to Met) in the rpsJ gene (rpsJ1) encoding ribosomal protein S10. Moreover, the identical mutation was found in six distinct tetracycline-resistant clinical isolates in which the MIC of tetracycline was ≥2 μg/ml. Site-saturation mutagenesis of the codon for Val-57 identified two other amino acids (Leu and Gln) that conferred identical levels of resistance as the Met-57 mutation. The mutation maps to the vertex of a loop in S10 that is near the aminoacyl-tRNA site in the structure of the 30S ribosomal subunit from Thermus thermophilus, and the residue equivalent to Val-57 in T. thermophilus S10, Lys-55, is within 8 to 9 Å of bound tetracycline. These data suggest that large noncharged amino acids alter the rRNA structure near the tetracycline-binding site, leading to a lower affinity of the antibiotic.

scholarly journals Molecular Basis of Ca2+ Activation of the Mouse Cardiac Ca2+ Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 118 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pin Li ◽  
S.R. Wayne Chen
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Single Channel ◽  
Single Point ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Single Channels ◽  
Wild Type ◽  
Structural And Functional Properties

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

scholarly journals Robotic QM/MM-driven maturation of antibody combining sites

2016 ◽  
Vol 2 (10) ◽  
pp. e1501695 ◽  
Author(s):  
Ivan V. Smirnov ◽  
Andrey V. Golovin ◽  
Spyros D. Chatziefthimiou ◽  
Anastasiya V. Stepanova ◽  
Yingjie Peng ◽  
...  
Keyword(s):  
Immune Response ◽  
In Vitro Selection ◽  
Single Point ◽  
Combinatorial Libraries ◽  
Single Point Mutation ◽  
Wild Type ◽  
Selection Of ◽  
Maturation Function

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

scholarly journals The Cholesterol Requirement for Sindbis Virus Entry and Exit and Characterization of a Spike Protein Region Involved in Cholesterol Dependence

1999 ◽  
Vol 73 (5) ◽  
pp. 4272-4278 ◽  
Author(s):  
Yanping E. Lu ◽  
Todd Cassese ◽  
Margaret Kielian
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Virus Entry ◽  
Single Point ◽  
Endocytic Pathway ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Entry And Exit ◽  
Wild Type

ABSTRACT Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV,srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had thesrf-3 amino acid sequence from E1 position 224 to 235 showed increases of ∼100-fold in infection and ∼250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGMexit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.

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