Molecular cloning and in silico characterization of two alpha-like neurotoxins and one metalloproteinase from the maxilllipeds of the centipede Scolopendra subspinipes mutilans

Abstract Aims In order to shed light of characterizations of centipede Scolopendra subspinipes mutilans venom, a two novel full-lengths of alpha-like-neurotoxin and one metalloproteinase cDNAs derived from the maxilllipeds RNA of centipede S. subspinipes mutilans were isolated, and, respectively, named as SsuTA1, SsuTA2 and SsuMPs. Materials and methods The SsuTA1, SsuTA2 and SsuMPs were cloned from the S. subspinipes mutilans using the rapid amplification of cDNA ends methods. Results In the current study, SsuTA1 and SsuTA2 were, respectively, composed of 82 amino acid residues and 106 amino acid residues. Deduced protein sequence of SsuTA1 shared high homology with that of SsuTA2, one major difference was the C-terminal 24-residue extension in SsuTA2. An abundance of cysteine residues and several adjacent beta-sheets were observed in SsuTA1 and SsuTA2. SsuMPs had 594 amino acid residues containing with a molecular mass of 68.29 kDa. The primary sequence analysis indicated that the SsuMPs contains a zinc-binding motif (HEIGHSLGLAHS) and methionine-turn motif (YIM). Phylogenetic analysis revealed early divergence and independent evolution of SsuTA1 and SsuTA2 from other α-neurotoxins. Conclusion The results suggested that centipede S. subspinipes mutilans is an ancient member of venomous arthropods, but its venom exhibits novel scenario.

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Structural Modelling and in-silico characterization of a Novel Thermophilic β-amylase from Clostridium thermosulfuregenes

Biotechnology Journal International ◽

10.9734/bji/2021/v25i430144 ◽

2021 ◽

pp. 1-22

Author(s):

Shafiqa Nayel ◽

Mohd Shahir Shamsir ◽

Farid Ahmad Danishfar

Keyword(s):

Amino Acid ◽

Binding Sites ◽

Hydrolytic Enzyme ◽

Beta Sheets ◽

Amino Acid Residues ◽

Structural Domains ◽

Structural Conformation ◽

In Silico Characterization

β-amylase is a hydrolytic enzyme that is involved in breaking down starch and producing energy. Since the discovery of β-amylase, it has been applied in various applications especially in the food industry. In this study, a novel β-amylase from Clostridium thermosuluregen, a thermophilic anaerobic bacterium that ferments its extracellular emulsion to ethanol at 62 ℃ was modelled and studied using bioinformatics tools and compared with B. cereus β-amylases that functions at mesophilic conditions. The results showed that the overall structural conformations, secondary structures, and important residues involved in active and binding sites were identified in both proteins. The results revealed that the modelled β-amylase of C. thermosulfuregen is very similar with respect to the global conformation, location of active and binding sites. Both proteins showed identical structural domains with the thermophilic variant possessing a high percentage of hydrophobic amino acid residues, polar amino acid residues, and differences in secondary composition such as loops and beta sheets as the potential evolutionary thermal adaptations that make it stable enzyme that functions up to 70 ℃. The results suggest that the thermal stability are not dependent on one single unique mechanism and may use one or a combination of the mechanisms to sustain its structural conformation at a higher operating temperature. Overall, considering the common properties of this modelled protein with the β-amylase of B. cereus, it can be assumed that if the β-amylase of C. thermosulfuregen were expressed in-vitro, it would produce a stable protein that possesses the hydrolysis function for C. thermosulfuregen to break down the starch and sugar formation.

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Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽

10.1186/s12864-021-07798-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhongying Wang ◽

Qixuan Wang ◽

Hao Wu ◽

Zhiwu Huang

Keyword(s):

Amino Acid ◽

Inner Ear ◽

Hair Cells ◽

Rana Catesbeiana ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Rna Seq ◽

American Bullfrog ◽

Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Journal of Bacteriology ◽

10.1128/jb.186.15.4885-4893.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4885-4893 ◽

Cited By ~ 154

Author(s):

Takane Katayama ◽

Akiko Sakuma ◽

Takatoshi Kimura ◽

Yutaka Makimura ◽

Jun Hiratake ◽

...

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Amino Acid Residues ◽

Gene Encoding ◽

Sugar Chain ◽

Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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Zn2+Ions Selectively Induce Antimicrobial Salivary Peptide Histatin-5 To Fuse Negatively Charged Vesicles. Identification and Characterization of a Zinc-Binding Motif Present in the Functional Domain†

Biochemistry ◽

10.1021/bi990212c ◽

1999 ◽

Vol 38 (30) ◽

pp. 9626-9633 ◽

Cited By ~ 51

Author(s):

Sonia Melino ◽

Stefano Rufini ◽

Marco Sette ◽

Roberto Morero ◽

Alessandro Grottesi ◽

...

Keyword(s):

Zinc Binding ◽

Binding Motif ◽

Functional Domain ◽

Histatin 5 ◽

Salivary Peptide ◽

Identification And Characterization ◽

Zinc Binding Motif

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Identification and in silico characterization of hemocyanin γ-type subunit protein in Vibrio harveyi infected freshwater prawn, Macrobrachium rosenbergii

Journal of Environmental Biology ◽

10.22438/jeb/42/1/mrn-1608 ◽

2021 ◽

Vol 42 (1) ◽

pp. 14-23

Author(s):

B.B. Patnaik ◽

◽

S. Baliarsingh ◽

S. Sahoo ◽

J.M. Chung ◽

...

Keyword(s):

Amino Acid ◽

In Silico ◽

Vibrio Harveyi ◽

Macrobrachium Rosenbergii ◽

Full Length ◽

Freshwater Prawn ◽

Amino Acid Residues ◽

Subunit Protein ◽

In Silico Tools

Aim: Identification of full-length ORF of hemocyanin subunit-1 (Mr_HC_1) from the hepatopancreas transcriptome of freshwater prawn, Macrobrachium rosenbergii infected with Vibrio harveyi and characterization of its sequence and structure by in silico tools and softwares. Methodology: Illumina HiSeq and de novo assembled unigenes were scanned against PANM-DB to screen Mr_HC_1. FGENESH gene prediction and SMART programs were used to predict the ORF region. Subsequently, Clustal X2 and MEGA in-silico tools were used to understand the sequence relatedness and evolutionary status of Mr_HC_1. Structural prediction was performed by SWISS-MODEL and Ramachandran plot modeling programs Results: The full-length ORF was 1983 bp in length encoding a polypeptide of 661 amino acid residues. Mr_HC_1 showed a putative signal peptide of 21 amino acid residues at the N-terminus and three hemocyanin domains. Homology analysis of Mr_HC_1 amino acid sequence confirms maximum identity to M. nipponense hemocyanin subunit-1 (Mn_HC_1). Phylogenetic analysis showed that Mr_HC_1 is more closely related to the hemocyanin γ-type subunit of freshwater shrimps. Homology modeling of Mr_HC_1 showed homo-hexameric protein containing 12 copper ions. With a QMEAN score of -3.33 and model-template sequence identity of 59.15%, the predicted model of Mr_HC_1 is convincing Interpretation: This study characterizes the hemocyanin γ-type subunit protein of freshwater prawn, M. rosenbergii for future studies on host defense mechanisms.

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