Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

Download Full-text

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19841846 ◽

1984 ◽

Vol 49 (8) ◽

pp. 1846-1853 ◽

Cited By ~ 4

Author(s):

Karel Hauzer ◽

Tomislav Barth ◽

Linda Servítová ◽

Karel Jošt

Keyword(s):

Amino Acids ◽

Aspartic Acid ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Relative Molecular Mass ◽

Enzyme Molecule ◽

Thiol Compounds ◽

Modified Method ◽

N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

Download Full-text

Avoided motifs: short amino acid strings missing from protein datasets

Biological Chemistry ◽

10.1515/hsz-2020-0383 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Pablo Mier ◽

Miguel A. Andrade-Navarro

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Function ◽

Large Protein ◽

New Approach ◽

Cellular Context ◽

Human Proteins ◽

Context Specific ◽

Protein Datasets

Abstract According to the amino acid composition of natural proteins, it could be expected that all possible sequences of three or four amino acids will occur at least once in large protein datasets purely by chance. However, in some species or cellular context, specific short amino acid motifs are missing due to unknown reasons. We describe these as Avoided Motifs, short amino acid combinations missing from biological sequences. Here we identify 209 human and 154 bacterial Avoided Motifs of length four amino acids, and discuss their possible functionality according to their presence in other species. Furthermore, we determine two Avoided Motifs of length three amino acids in human proteins specifically located in the cytoplasm, and two more in secreted proteins. Our results support the hypothesis that the characterization of Avoided Motifs in particular contexts can provide us with information about functional motifs, pointing to a new approach in the use of molecular sequences for the discovery of protein function.

Download Full-text

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o84-038 ◽

1984 ◽

Vol 62 (5) ◽

pp. 276-279 ◽

Cited By ~ 1

Author(s):

C. H. Lin ◽

W. Chung ◽

K. P. Strickland ◽

A. J. Hudson

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Enzymatic Synthesis ◽

Gel Filtration ◽

Deae Cellulose ◽

Aromatic Amino Acids ◽

Adenosylmethionine Synthetase ◽

Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

Download Full-text

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im [corrected] associated with a sulI-type integron.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.2.314 ◽

1997 ◽

Vol 41 (2) ◽

pp. 314-318 ◽

Cited By ~ 32

Author(s):

E Hannecart-Pokorni ◽

F Depuydt ◽

L de wit ◽

E van Bossuyt ◽

J Content ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Resistance Gene ◽

Citrobacter Freundii ◽

Gram Negative ◽

Gene Environment ◽

Insert Region ◽

Klebsiella Spp

The amikacin resistance gene aac(6')-Im [corrected] from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron. The sequence of this aac(6')-Im [corrected] gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C. Tenover, D. Filpula, K.L. Phillips, and J. J. Plorde, J. Bacteriol. 170:471-473, 1988). By using PCR, the aac(6')-Im [corrected] gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli. PCR mapping of the aac(6')-Im [corrected] gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

Download Full-text

Genetic characterization and phylogenetic study of Indonesian indigenous catfish based on mitochondrial cytochrome B gene

Veterinary World ◽

10.14202/vetworld.2020.96-103 ◽

2020 ◽

Vol 13 (1) ◽

pp. 96-103

Author(s):

Dorothea Vera Megarani ◽

Herjuno Ari Nugroho ◽

Zahrah Prawita Andarini ◽

Yura Dwi Risa B. R. Surbakti ◽

Rini Widayanti

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cytochrome B ◽

Genetic Characterization ◽

Phylogenetic Study ◽

Cyt B ◽

Phylogenetic Structure ◽

Mitochondrial Cytochrome B ◽

Sperata Seenghala ◽

Cyt B Gene

Aim: This study aimed to determine the genetic characterization and phylogenetic structure of Indonesian indigenous catfish using cytochrome B (Cyt B) sequences. Materials and Methods: The genomes of 26 catfishes caught from nine rivers from nine different geographical locations around Indonesia were analyzed. The tissue isolation method was used to isolate the total genome of the fishes. Furthermore, polymerase chain reaction was done to amplify the mtDNA Cyt B using the CytBF and CytBR primers. Following sequencing, the analysis of genetic variation and the phylogenetic relationship was performed using MEGA version X software. Results: Cyt B gene sequencing attained a total of 1139 nucleotides encrypting 379 amino acids for all samples. The ClustalW alignment program using MEGA X software revealed 395 substituted nucleotides, which then translated into 63 amino acid variation sites among all 26 samples. No amino acids in catfish BB were different compared to catfish PM, MP, and KR2,3. Catfish MS had one modified amino acid; KR1 and KS had two different amino acids; BF had 38 different amino acids; EM had 31 different amino acids; and BSBJ had 26 different amino acids compared to catfish BB. The most significant alteration of amino acids was between catfish EM and BF (49 amino acids). Conclusion: Indonesian catfish were divided into five clades based on the Cyt B gene. Samples KR and MP (Sumatra); MS and BB (Kalimantan); and PM (Java) were clustered with Hemibagrus nemurus and Hemibagrus wyckioides (Bagridae family). Samples from Kalimantan (KS) and one sample of KR (KR1) from Sumatra were clustered with Sperata seenghala and Hemibagrus spilopterus (Bagridae family). Samples from Java (BSBJ) were clustered with Pseudolais pleurotaenia (Pangasiidae family). Samples EM (Java) were together with Mystus cavasius (Bagridae family). Samples from West Papua were clustered with Potamosilurus latirostris (Ariidae family).

Download Full-text

Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

10.26434/chemrxiv.14206172.v1 ◽

2021 ◽

Author(s):

Hans Renata ◽

Emily Shimizu ◽

Christian Zwick

Keyword(s):

Amino Acid ◽

Building Block ◽

Solid Phase ◽

Functional Characterization ◽

Free Standing ◽

Substrate Specificities ◽

Total Turnover

We report the functional characterization of two iron- and a-ketoglutarate-dependent dioxygenases that are capable of hydroxylating free-standing glutamine at its C3 and C4 position respectively. In particular, the C4 hydroxylase, Q4Ox, catalyzes the reaction with approximately 4,300 total turnover numbers, facilitating synthesis of a solid-phase compatible building block and stereochemical elucidation at the C4 position of the hydroxylated product. This work will enable the development of novel synthetic strategies to prepare useful glutamine derivatives and stimulate further discoveries of new amino acid hydroxylases with distinct substrate specificities.<br>

Download Full-text

Urotensin receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

IUPHAR/BPS Guide to Pharmacology CITE ◽

10.2218/gtopdb/f65/2019.4 ◽

2019 ◽

Vol 2019 (4) ◽

Author(s):

Anthony P. Davenport ◽

Stephen A. Douglas ◽

Alain Fournier ◽

Adel Giaid ◽

Henry Krum ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurosecretory System ◽

Urotensin Ii ◽

High Sequence Identity ◽

Caudal Neurosecretory System ◽

Amino Acid Peptide ◽

Related Peptide ◽

Human Sequence ◽

Mature Mouse

The urotensin-II (U-II) receptor (UT, nomenclature as agreed by the NC-IUPHAR Subcommittee on the Urotensin receptor [26, 36, 89]) is activated by the endogenous dodecapeptide urotensin-II, originally isolated from the urophysis, the endocrine organ of the caudal neurosecretory system of teleost fish [7, 88]. Several structural forms of U-II exist in fish and amphibians. The goby orthologue was used to identify U-II as the cognate ligand for the predicted receptor encoded by the rat gene gpr14 [20, 62, 68, 70]. Human urotensin-II, an 11-amino-acid peptide [20], retains the cyclohexapeptide sequence of goby U-II that is thought to be important in ligand binding [53, 11]. This sequence is also conserved in the deduced amino-acid sequence of rat urotensin-II (14 amino-acids) and mouse urotensin-II (14 amino-acids), although the N-terminal is more divergent from the human sequence [19]. A second endogenous ligand for the UT has been discovered in rat [83]. This is the urotensin II-related peptide, an octapeptide that is derived from a different gene, but shares the C-terminal sequence (CFWKYCV) common to U-II from other species. Identical sequences to rat urotensin II-related peptide are predicted for the mature mouse and human peptides [32]. UT exhibits relatively high sequence identity with somatostatin, opioid and galanin receptors [89].

Download Full-text

Regiodivergent Biocatalytic Hydroxylation of L-Glutamine Facilitated by Characterization of Non-Heme Dioxygenases from Non-Ribosomal Peptide Biosyntheses

10.26434/chemrxiv.14206172 ◽

2021 ◽

Author(s):

Hans Renata ◽

Emily Shimizu ◽

Christian Zwick

Keyword(s):

Amino Acid ◽

Building Block ◽

Solid Phase ◽

Functional Characterization ◽

Free Standing ◽

Substrate Specificities ◽

Total Turnover

Download Full-text

In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

10.21203/rs.3.rs-366314/v2 ◽

2021 ◽

Author(s):

Amrutha Bindu ◽

Lakshmi Devi

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Lactobacillus Plantarum ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Proteinase K ◽

Kinetic Assay ◽

Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

Download Full-text