scholarly journals Structure Prediction of a Thermostable SR74 α-Amylase from Geobacillus stearothermophilus Expressed in CTG-Clade Yeast Meyerozyma guilliermondii Strain SO

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1059
Author(s):  
Si Jie Lim ◽  
Noor Dina Muhd Noor ◽  
Abu Bakar Salleh ◽  
Siti Nurbaya Oslan
Keyword(s):  
Structure Prediction ◽  
In Silico Analysis ◽  
Biochemical Properties ◽  
Amino Acid Residues ◽  
Geobacillus Stearothermophilus ◽  
Multiple Sequence ◽  
Meyerozyma Guilliermondii ◽  
Glycosidic Bonds ◽  
And Function ◽  
Hydrolysis Of

α-amylase which catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch have frequently been cloned into various microbial workhorses to yield a higher recombinant titer. A thermostable SR74 α-amylase from Geobacillus stearothermophilus was found to have a huge potential in detergent industries due to its thermostability properties. The gene was cloned into a CTG-clade yeast Meyerozyma guilliermondii strain SO. However, the CUG ambiguity present in the strain SO has possibly altered the amino acid residues in SR74 amylase wild type (WT) encoded by CUG the codon from the leucine to serine. From the multiple sequence alignment, six mutations were found in recombinant SR74 α-amylase (rc). Their effects on SR74 α-amylase structure and function remain unknown. Herein, we predicted the structures of the SR74 amylases (WT and rc) using the template 6ag0.1.A (PDB ID: 6ag0). We sought to decipher the possible effects of CUG ambiguity in strain SO via in silico analysis. They are structurally identical, and the metal triad (CaI–CaIII) might contribute to the thermostability while CaIV was attributed to substrate specificity. Since the pairwise root mean square deviation (RMSD) between the WT and rc SR74 α-amylase was lower than the template, we suggest that the biochemical properties of rc SR74 α-amylase were better deduced from its WT, especially its thermostability.

Molecular Cloning and Structural Insights into pectin lyase proteins from different strains of Fusarium

2020 ◽  
Vol 17 ◽  
Author(s):  
Sangeeta Yadav ◽  
Gautam Anand ◽  
Vinay K Singh ◽  
Dinesh Yadav
Keyword(s):  
Structure Prediction ◽  
Disulfide Bonds ◽  
3D Structure ◽  
Chemical Characterization ◽  
Structural Features ◽  
Docking Studies ◽  
Biochemical Properties ◽  
Pectin Lyase ◽  
Homology Search ◽  
Multiple Sequence

: Pectin lyaseis an industrially important enzymeof pectinase group that degrade pectin polymers forming 4,5-unsaturated oligogalacturonides. Several fugal pectin lyase genes predominately from Aspergillus and Penicillium genera have been reported in the literature. Five pectin lyase genes were cloned from FusariumoxysporumMTCC1755, F.monoliforme var. subglutinansMTCC2015, FusariumavneceumMTCC10572, and FusariumsolaniMTCC3004 using PCR approach. Pectin lyase genes and proteins were subjected to homology search, multiple sequence alignment, motif search, physio-chemical characterization, phylogenetic tree construction, 3D structure prediction and molecular docking. Many conserved amino acids were found at several positions in all the pectin lyase proteins. Phylogenetic analysis of these proteins alongwith other pectinases revealed two major clusters representing members of lyases and hydrolases. In-silico characterization revealed pectin lyase proteins to be highly stable owing to the presence of disulfide bonds in their structure. Molecular weight and pI of these proteins were in the range 14.4 to 25.1 kDa and 4.47-9.39 respectively. Pectin lyase proteins from different Fusariumstrains were very much similar in their structural features and biochemical properties which might be due to their similarity on the primary sequence. Docking studies revealed that electrostatic forces, vander Waal and hydrogen bonds are the major interacting forces between the ligands and the enzyme. This might be accountable for comparatively higher and better activity of pectin lyase against galacturonic acid as compared to α-D-galactopyranuronic acid, galactofuranuronicacid and galactopyranuronate. Aspartate, tyrosine and tryptophan residues in the active site of the enzyme are responsible for ligand binding.

Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum

Parasitology ◽  
2011 ◽  
Vol 138 (6) ◽  
pp. 682-690 ◽  
Author(s):  
J.-M. KANG ◽  
H.-L. JU ◽  
W.-M. SOHN ◽  
B.-K. NA
Keyword(s):  
Amino Acid ◽  
Cryptosporidium Parvum ◽  
Metal Binding ◽  
Drug Targets ◽  
Leucine Aminopeptidase ◽  
Biochemical Properties ◽  
Site Formation ◽  
Amino Acid Residues ◽  
Multiple Sequence ◽  
Binding Characteristics

SUMMARYLeucine aminopeptidases (LAPs) are a group of metalloexopeptidases that catalyse the sequential removal of amino acids from the N-termini of polypeptides or proteins. They play an important role in regulating the balance between catabolism and anabolism in living cells. LAPs of apicomplexa parasitic protozoa have been intensively investigated due to their crucial roles in parasite biology as well as their potentials as drug targets. In this study, we identified an M17 leucine aminopeptidase of Cryptosporidium parvum (CpLAP) and characterized the biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of CpLAP with those of other organisms revealed that typical amino acid residues essential for metal binding and active-site formation in M17 LAPs were well conserved in CpLAP. Recombinant CpLAP shared similar biochemical properties such as optimal pH, stability at neutral pHs, and metal-binding characteristics with other characterized LAPs. The enzyme showed a marked preference for Leu and its activity was effectively inhibited by bestatin. These results collectively suggest that CpLAP is a typical member of the M17 LAP family and may play an important role in free amino acid regulation in the parasite.

scholarly journals fastMSA: Accelerating Multiple Sequence Alignment with Dense Retrieval on Protein Language

2021 ◽  
Author(s):  
Liang Hong ◽  
Siqi Sun ◽  
Liangzhen Zheng ◽  
Qingxiong Tan ◽  
Yu Li
Keyword(s):  
Protein Structure ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Structure Prediction ◽  
Structure And Function ◽  
Sequence Alignments ◽  
Protein Structure And Function ◽  
Multiple Sequence ◽  
Multiple Sequence Alignments ◽  
And Function

Evolutionarily related sequences provide information for the protein structure and function. Multiple sequence alignment, which includes homolog searching from large databases and sequence alignment, is efficient to dig out the information and assist protein structure and function prediction, whose efficiency has been proved by AlphaFold. Despite the existing tools for multiple sequence alignment, searching homologs from the entire UniProt is still time-consuming. Considering the success of AlphaFold, foreseeably, large- scale multiple sequence alignments against massive databases will be a trend in the field. It is very desirable to accelerate this step. Here, we propose a novel method, fastMSA, to improve the speed significantly. Our idea is orthogonal to all the previous accelerating methods. Taking advantage of the protein language model based on BERT, we propose a novel dual encoder architecture that can embed the protein sequences into a low-dimension space and filter the unrelated sequences efficiently before running BLAST. Extensive experimental results suggest that we can recall most of the homologs with a 34-fold speed-up. Moreover, our method is compatible with the downstream tasks, such as structure prediction using AlphaFold. Using multiple sequence alignments generated from our method, we have little performance compromise on the protein structure prediction with much less running time. fastMSA will effectively assist protein sequence, structure, and function analysis based on homologs and multiple sequence alignment.

Computational Analysis of Therapeutic Enzyme Uricase from Different Source Organisms

2020 ◽  
Vol 17 (1) ◽  
pp. 59-77
Author(s):  
Anand Kumar Nelapati ◽  
JagadeeshBabu PonnanEttiyappan
Keyword(s):  
Uric Acid ◽  
Amino Acid ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Protein Sequences ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Multiple Sequence ◽  
Physiochemical Properties ◽  
Pharmaceutical Industries

Background:Hyperuricemia and gout are the conditions, which is a response of accumulation of uric acid in the blood and urine. Uric acid is the product of purine metabolic pathway in humans. Uricase is a therapeutic enzyme that can enzymatically reduces the concentration of uric acid in serum and urine into more a soluble allantoin. Uricases are widely available in several sources like bacteria, fungi, yeast, plants and animals.Objective:The present study is aimed at elucidating the structure and physiochemical properties of uricase by insilico analysis.Methods:A total number of sixty amino acid sequences of uricase belongs to different sources were obtained from NCBI and different analysis like Multiple Sequence Alignment (MSA), homology search, phylogenetic relation, motif search, domain architecture and physiochemical properties including pI, EC, Ai, Ii, and were performed.Results:Multiple sequence alignment of all the selected protein sequences has exhibited distinct difference between bacterial, fungal, plant and animal sources based on the position-specific existence of conserved amino acid residues. The maximum homology of all the selected protein sequences is between 51-388. In singular category, homology is between 16-337 for bacterial uricase, 14-339 for fungal uricase, 12-317 for plants uricase, and 37-361 for animals uricase. The phylogenetic tree constructed based on the amino acid sequences disclosed clusters indicating that uricase is from different source. The physiochemical features revealed that the uricase amino acid residues are in between 300- 338 with a molecular weight as 33-39kDa and theoretical pI ranging from 4.95-8.88. The amino acid composition results showed that valine amino acid has a high average frequency of 8.79 percentage compared to different amino acids in all analyzed species.Conclusion:In the area of bioinformatics field, this work might be informative and a stepping-stone to other researchers to get an idea about the physicochemical features, evolutionary history and structural motifs of uricase that can be widely used in biotechnological and pharmaceutical industries. Therefore, the proposed in silico analysis can be considered for protein engineering work, as well as for gout therapy.

In Silico Study of Potential Cross-Kingdom Plant MicroRNA Based Regulation in Chronic Myeloid Leukemia

2020 ◽  
Vol 17 (2) ◽  
pp. 125-132
Author(s):  
Marjanu Hikmah Elias ◽  
Noraziah Nordin ◽  
Nazefah Abdul Hamid
Keyword(s):  
Targeted Therapy ◽  
In Silico ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Target Prediction ◽  
In Silico Analysis ◽  
Microrna Target ◽  
Good Target

Background: Chronic Myeloid Leukaemia (CML) is associated with the BCRABL1 gene, which plays a central role in the pathogenesis of CML. Thus, it is crucial to suppress the expression of BCR-ABL1 in the treatment of CML. MicroRNA is known to be a gene expression regulator and is thus a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the microRNAs from edible plants targeting the 3’ Untranslated Region (3’UTR) of BCR-ABL1. Methods: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were used to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. The MiRBase database was used to validate the species of plants expressing the miRNAs. The RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction, respectively. Results: In silico analyses revealed that cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b show binding conformity with strong molecular interaction towards 3’UTR region of BCR-ABL1. However, only cpa-miR- 8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these microRNAs post-transcriptionally inhibit the BCRABL1 gene and thus could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out to determine the ability of these miRNAs to form the basis for targeted therapy for CML.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

Bioinformatics analysis of bacteriophage and prophage endolysin domains

Biologia ◽  
2014 ◽  
Vol 69 (5) ◽  
Author(s):  
Barbora Vidová ◽  
Zuzana Šramková ◽  
Lenka Tišáková ◽  
Michaela Oravkinová ◽  
Andrej Godány
Keyword(s):  
Antimicrobial Properties ◽  
In Silico Analysis ◽  
Multiple Sequence ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Multiresistant Bacteria ◽  
Or Gene ◽  
The Individual ◽  
Conserved Amino Acids ◽  
Sequence Types

AbstractEndolysins as a class of antibacterial enzymes are expected to become a very useful tool for many purposes to control spreading of, e.g., multiresistant bacteria in different environments. Their antimicrobial properties could be broadened or altered by mutagenesis, domain swapping or gene shuffling. Therefore, the specific designing of endolysins to achieve their desired properties is challenging. This work is focused on the in silico analysis of protein domains presence in sequences of phage and prophage endolysins, followed by the study of variety of domain combinations in the individual endolysin types. The multiple sequence alignment of endolysin sequences revealed the recognition of sequence types with typical domain arrangement and conserved amino acids, divided according to the target substrate in bacterial cell walls. The five protein families of catalytic domains are specifically occurring in dependence of bacterial Gram-type. The presence, types and numbers of binding domains within endolysin sequences were also studied. The obtained results enable a more targeted design of endolysins with required antimicrobial properties.

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

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
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).

scholarly journals Stability of Methylphenidate under Various pH Conditions in the Presence or Absence of Gut Microbiota

2021 ◽  
Vol 14 (8) ◽  
pp. 733
Author(s):  
Julia Aresti-Sanz ◽  
Markus Schwalbe ◽  
Rob Rodrigues Pereira ◽  
Hjalmar Permentier ◽  
Sahar El Aidy
Keyword(s):  
Gut Microbiota ◽  
In Silico Analysis ◽  
Intestinal Bacteria ◽  
Inactive Form ◽  
High Interindividual Variability ◽  
Ritalinic Acid ◽  
Ph Conditions ◽  
Small Intestinal ◽  
The Stability ◽  
Hydrolysis Of

Methylphenidate is one of the most widely used oral treatments for attention-deficit/hyperactivity disorder (ADHD). The drug is mainly absorbed in the small intestine and has low bioavailability. Accordingly, a high interindividual variability in terms of response to the treatment is known among ADHD patients treated with methylphenidate. Nonetheless, very little is known about the factors that influence the drug’s absorption and bioavailability. Gut microbiota has been shown to reduce the bioavailability of a wide variety of orally administered drugs. Here, we tested the ability of small intestinal bacteria to metabolize methylphenidate. In silico analysis identified several small intestinal bacteria to harbor homologues of the human carboxylesterase 1 enzyme responsible for the hydrolysis of methylphenidate in the liver into the inactive form, ritalinic acid. Despite our initial results hinting towards possible bacterial hydrolysis of the drug, up to 60% of methylphenidate is spontaneously hydrolyzed in the absence of bacteria and this hydrolysis is pH-dependent. Overall, our results indicate that the stability of methylphenidate is compromised under certain pH conditions in the presence or absence of gut microbiota.

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