Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis

2021 ◽  
Author(s):  
Maribel Cayetano-Cruz ◽  
Luis A Caro-Gómez ◽  
Miguel Plascencia-Espinosa ◽  
Alejandro Santiago-Hernández ◽  
Claudia G Benítez-Cardoza ◽  
...  
Keyword(s):  
Simulation Analysis ◽  
Fold Increase ◽  
Md Simulations ◽  
Biochemical Properties ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Optimal Activity ◽  
Upward Shift ◽  
Structure Changes ◽  
Cellulomonas Uda

Abstract Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 ºC in the optimum temperature, and an upward shift of one unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 ºC, compared to that displayed by Xyn11A-WT. Molecular dynamics (MD) simulations of Xyn11A-WT and Xyn11A-N9Y suggest the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76040-76047 ◽  
Author(s):  
Zhenya Chen ◽  
Ye Li ◽  
Yue Feng ◽  
Liang Chen ◽  
Qipeng Yuan
Keyword(s):  
Active Site ◽  
Simulation Analysis ◽  
Docking Simulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Molecular Docking Simulation ◽  
Activity Enhancement ◽  
First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

scholarly journals Double Mutations in Succinate Dehydrogenase Are Involved in SDHI Resistance in Corynespora cassiicola

2022 ◽  
Vol 10 (1) ◽  
pp. 132
Author(s):  
Bingxue Sun ◽  
Guangxue Zhu ◽  
Xuewen Xie ◽  
Ali Chai ◽  
Lei Li ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Point Mutation ◽  
Single Point ◽  
Resistance Management ◽  
Site Directed Mutagenesis ◽  
Single Point Mutation ◽  
Single Mutation ◽  
Corynespora Cassiicola ◽  
Double Mutation ◽  
Mutation Analyses

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

scholarly journals An integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation

2021 ◽  
Author(s):  
Nadendla EswarKumar ◽  
Cheng-Han Yang ◽  
Sunilkumar Tewary ◽  
Yi-Qi Yeh ◽  
Hsiao-Ching Yang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complex ◽  
Tyrosine Phosphatase ◽  
Complex Structure ◽  
Protein Crystallography ◽  
Md Simulations ◽  
Integrative Approach ◽  
Single Mutation ◽  
Protein Protein Interactions ◽  
Transient Nature

AbstractProtein tyrosine phosphatase: phospho-protein complex structure determination, which requires to understand how specificity is achieved at the protein level remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we established an integrative workflow involving protein crystallography, SAXS and pTyr-tailored MD simulations to reveal the complex formed between rPTPεD1 and phospho-SrcKD, revealing transient protein–protein interactions distal to the active site. To support our finding, we determined the associate rate between rPTPεD1 and phospho-SrcKD and showed that a single mutation on rPTPεD1 disrupts this transient interaction, resulting in the reduction of association rate and activity. Our simulations suggest that rPTPεD1 employs a binding mechanism involving conformational change prior to the engagement of cSrcKD. This integrative approach is applicable to other PTP: phospho-protein complex determination and is a general approach for elucidating transient protein surface interactions.

scholarly journals SHV-16, a β-Lactamase with a Pentapeptide Duplication in the Omega Loop

2001 ◽  
Vol 45 (9) ◽  
pp. 2480-2485 ◽  
Author(s):  
Corinne Arpin ◽  
Roger Labia ◽  
Catherine Andre ◽  
Cécile Frigo ◽  
Zoubida El Harrif ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Donor Cell ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Resistance Pattern ◽  
E Coli ◽  
Omega Loop ◽  
Low Levels ◽  
Encoding Gene ◽  
Level Resistance

ABSTRACT A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 μg/ml), ticarcillin (MIC of 512 μg/ml), and ceftazidime (MIC of 128 μg/ml) and susceptible to all other β-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum β-lactamase (ESBL). Transconjugants inEscherichia coli were obtained at low levels (10−7 per donor cell) and exhibited a similar β-lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 μg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR usingbla SHV-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla SHV-1 orbla SHV-16 in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.

scholarly journals Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

2019 ◽  
Vol 20 (6) ◽  
pp. 1410 ◽  
Author(s):  
Xiaohua Wang ◽  
Kai Song ◽  
Yang Li ◽  
Ling Tang ◽  
Xin Deng
Keyword(s):  
Molecular Dynamics ◽  
Green Fluorescent Protein ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Hydrophobic Interactions ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Functional Protein ◽  
Green Fluorescent

Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.

scholarly journals Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

2019 ◽  
Vol 295 (2) ◽  
pp. 597-609 ◽  
Author(s):  
Taeho Kim ◽  
Peter J. Stogios ◽  
Anna N. Khusnutdinova ◽  
Kayla Nemr ◽  
Tatiana Skarina ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Thermotoga Maritima ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Bacillus Halodurans ◽  
Active Site Residues ◽  
Carbon Carbon Bond ◽  
Condensation Activity

Carbon–carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli. The crystal structures of BH1352 and TM1559 at 1.40–2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

scholarly journals Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jun Li ◽  
Haihong Hao ◽  
Menghong Dai ◽  
Heying Zhang ◽  
Jianan Ning ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pumps ◽  
Genetic Alterations ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Genetic Characteristics ◽  
Content Type ◽  
E Coli ◽  
Low Level ◽  
High Level

ABSTRACT This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 μg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 μg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

scholarly journals Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

2002 ◽  
Vol 76 (4) ◽  
pp. 1932-1943 ◽  
Author(s):  
Thomas P. Monath ◽  
Juan Arroyo ◽  
Inessa Levenbook ◽  
Zhen-Xi Zhang ◽  
John Catalan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lethal Dose ◽  
Vero Cells ◽  
Hinge Region ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Safety Testing ◽  
Important Region ◽  
Flavivirus Vaccine ◽  
Antibody Levels

ABSTRACT A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met→Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans.

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