Subtypes of NanS-p Sialate O-Acetylesterase Encoded by Stx2a Bacteriophages

Shiga toxin (Stx)-producing Escherichia coli strains are foodborne pathogens that can cause severe human diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Stxs are encoded by bacteriophages (Stx phages) which show remarkable variations in genome composition and harbour several genes of unknown function. Recently, a gene encoding a sialate O-acetylesterase (NanS-p) was identified in some relevant Stx2a phages and it was suggested that it could provide advantages for bacterial growth in the gut. The aim of this study was to analyse the presence and sequence of nanS-p genes in available Stx2a phage genomes. A total of 59 DNA sequences of Stx2a phages were extracted from the NCBI GenBank database with the BLAST program using the stx2a sequence from phage 933W as a query sequence, either as complete phage genomes (45) or from bacterial genomes by subsequent analysis with the PHASTER web server (14). Comparative analysis revealed that nanS-p was located downstream of stx2a in all genomes. Twenty different amino acid sequences of NanS-p were identified. Specifically, catalytic esterase domains showed only 11 possible sequences, with differences mainly observed in nine amino acid positions. Sequences corresponding to the N-terminal domain (DUF1737) showed three possible sequences, two of them closely related, while the C-terminal domain was highly variable, with four groups with structural differences. Since sialate O-acetylesterase activity has been determined from particular Stx2a phages, new studies are necessary to evaluate if the NanS-p subtypes identified in the present study also differ in their biological activity.

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Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity

Microbiology ◽

10.1099/mic.0.27878-0 ◽

2005 ◽

Vol 151 (7) ◽

pp. 2343-2351 ◽

Cited By ~ 20

Author(s):

Anne Dhalluin ◽

Ingrid Bourgeois ◽

Martine Pestel-Caron ◽

Emilie Camiade ◽

Gregory Raux ◽

...

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Clostridium Difficile ◽

Catalytic Domain ◽

Sequence Similarity ◽

Amino Acid Sequences ◽

Cellular Growth ◽

Peptidoglycan Hydrolase ◽

Gene Encoding ◽

Terminal Domain

A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.

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Using bioinformatic tools in exploited gene encoding enzyme to decompose lignocellulose from metagenome of free - living bacteria in the gut of the lower termite Coptotermes gestroi

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/14/1/9290 ◽

2016 ◽

Vol 14 (1) ◽

pp. 39-47

Author(s):

Nguyễn Minh Giang ◽

Đỗ Thị Huyền ◽

Trương Nam Hải

Keyword(s):

Amino Acid ◽

Melting Temperature ◽

Dna Sequences ◽

Amino Acid Sequences ◽

Open Reading Frames ◽

Gene Encoding ◽

Conserved Domains ◽

Bioinformatic Tools ◽

Coptotermes Gestroi ◽

Hydrolysis Of

Microbial metagenome DNA in the guts of Coptotermes gestroi has been extracted and sequenced by metagenomic techniques. In previous studies, we acquired and sequenced more than 5 Gb of DNA metagenome DNA of the termite gut microbiota by next-generation sequencing (Illumina). Software MGA (MetaGeneAnnotator) exploited 125.431 open reading frames with 8508 ORFs related to carbohydrate metabolism, including 587 ORFs coding for enzymes involved in the hydrolysis of lignocellulose. We identified software to reliably predict function, structure and characteristics of proteins corresponding to DNA sequences encoding alkaline enzymes from the metgenome of C gestroi. The online software Alcapred was used to predict alkaline enzymes, Blastp to predict conserved domains of amino acid sequences deduced from ORFs, Phyre2 to predict the three dimentional structure and substrate binding site of the enzymes, TBI to predict melting temperature of the enzyme. We identified 6 ORFs encoding alkaline cellulases (GL0101308, GL0038126) or alkaline hemicellulases (GL0120095, GL0074258, GL0112518, GL0067868). The amino acid sequences deduced from ORFs had 90% coverage and from 44% to 99% identity to the corresponding sequences in NCBI by BLASTP. All of them contained conserved domains with corresponding activities and binding sites of the enzyme to the substrate. The three dimentional structures of amino acid sequences were predicted by Phyre2 with reliability from 98% to 100% to the annotated activities. Among six selected amino acid sequences, two sequences of enzymes had the melting temperature above 65 ℃, three sequences had melting temperature from 55℃ to 65℃ and only one below 55℃.

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Purification and characterization of a uterine retinol-binding protein in the bitch

Biochemical Journal ◽

10.1042/bj3110407 ◽

1995 ◽

Vol 311 (2) ◽

pp. 407-415 ◽

Cited By ~ 9

Author(s):

W C Buhi ◽

I M Alvarez ◽

V M Shille ◽

M J Thatcher ◽

J P Harney ◽

...

Keyword(s):

Amino Acid ◽

Dna Sequences ◽

Binding Protein ◽

Amino Acid Content ◽

Amino Acid Sequences ◽

Retinol Binding Protein ◽

Total Amino Acid ◽

Major Protein ◽

Serum Retinol ◽

Two Dimensional Gel Electrophoresis

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

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Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.963-971.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 963-971

Author(s):

B Fenton ◽

J T Clark ◽

C M Khan ◽

J V Robinson ◽

D Walliker ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Amino Acid ◽

Surface Antigen ◽

Dna Sequences ◽

Amino Acid Sequences ◽

Merozoite Surface Antigen ◽

Parasite Plasmodium ◽

Genes Encoding ◽

Parasite Plasmodium Falciparum ◽

Group B

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.

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Alignment of Amino Acid and DNA Sequences of Human Proline-rich Proteins

Critical Reviews in Oral Biology & Medicine ◽

10.1177/10454411930040030501 ◽

1993 ◽

Vol 4 (3) ◽

pp. 287-292 ◽

Cited By ~ 12

Author(s):

D.L. Kauffman ◽

P.J. Keller ◽

A. Bennick ◽

M. Blum

Keyword(s):

Amino Acid ◽

Dna Sequences ◽

Sequence Data ◽

Gel Filtration ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Secreted Proteins ◽

Dna Encoding ◽

Protein Amino Acid ◽

Primary Gene

Human proline-rich proteins (PRPs) constitute a complex family of salivary proteins that are encoded by a small number of genes. The primary gene product is cleaved by proteases, thereby giving rise to about 20 secreted proteins. To determine the genes for the secreted PRPs, therefore, it is necessary to obtain sequences of both the secreted proteins and the DNA encoding these proteins. We have sequenced most PRPs from one donor (D.K.) and aligned the protein sequences with available DNA sequences from unrelated individuals. Partial sequence data have now been obtained for an additional PRP from D.K. named II-1. This protein was purified from parotid saliva by gel filtration and ion-exchange chromatography. Peptides were obtained by cleavage with trypsin, clostripain, and N-bromosuccinimide, followed by column chromatography. The peptides were sequenced on a gas-phase protein sequenator. Overlapping peptide sequences were obtained for most of II-1 and aligned with translated DNA sequences. The best fit was obtained with clones containing sequences for the allele PRB4" (Lyons et al., 1988). However, there was not complete identity of the protein amino acid sequence and the DNA-derived sequences, indicating that II-1 is not encoded by PRB4". Other PRPs isolated from D.K. also fail to conform to any DNA structure so far reported. This shows the need to obtain amino acid sequences and corresponding DNA sequences from the same person to assign genes for the PRPs and to determine the location of the postribosomal cleavage points in the primary translation product.

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Molecular Heterogeneity of the L-1 Metallo-β-Lactamase Family from Stenotrophomonas maltophilia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.5.1245 ◽

1998 ◽

Vol 42 (5) ◽

pp. 1245-1248 ◽

Cited By ~ 28

Author(s):

François Sanschagrin ◽

Julien Dufresne ◽

Roger C. Levesque

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Stenotrophomonas Maltophilia ◽

Amino Acid Sequences ◽

Molecular Heterogeneity ◽

Gene Encoding ◽

Class B

ABSTRACT We have determined the nucleotide sequence of the blaSgene encoding the carbapenem-hydrolyzing L-1 β-lactamase fromStenotrophomonas maltophilia GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc β-lactamases showed 88.6% identity with the L-1 enzyme fromS. maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.

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Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

Applied and Environmental Microbiology ◽

10.1128/aem.01101-10 ◽

2010 ◽

Vol 76 (17) ◽

pp. 5892-5901 ◽

Cited By ~ 51

Author(s):

Yoshikazu Shimada ◽

Setsuko Yasuda ◽

Masayuki Takahashi ◽

Takashi Hayashi ◽

Norihiro Miyazawa ◽

...

Keyword(s):

Amino Acid ◽

Enteric Bacteria ◽

Amino Acid Sequences ◽

Soy Isoflavones ◽

Cloning And Expression ◽

Gene Encoding ◽

Binding Motifs ◽

Reading Frame ◽

Cofactor Binding ◽

International Patent

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

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Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2824-2828.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2824-2828 ◽

Cited By ~ 2

Author(s):

Bettina Bäuerle ◽

Željko Cokesa ◽

Silvia Hofmann ◽

Paul-Gerhard Rieger

Keyword(s):

Amino Acid ◽

Agrobacterium Tumefaciens ◽

Heterologous Expression ◽

Bacterial Strain ◽

Amino Acid Sequences ◽

Complexing Agent ◽

Recombinant Enzymes ◽

Gene Encoding ◽

Degrading Bacteria

ABSTRACT Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl B and icl S, encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl B gene was chromosomal like the corresponding icl S gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.

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Characterization and Synergistic Interactions of Fibrobacter succinogenes Glycoside Hydrolases

Applied and Environmental Microbiology ◽

10.1128/aem.01037-07 ◽

2007 ◽

Vol 73 (19) ◽

pp. 6098-6105 ◽

Cited By ~ 38

Author(s):

Meng Qi ◽

Hyun-Sik Jun ◽

Cecil W. Forsberg

Keyword(s):

Amino Acid ◽

Synergistic Effect ◽

Genome Sequence ◽

Glycoside Hydrolase ◽

Amino Acid Sequences ◽

Glycoside Hydrolases ◽

Fibrobacter Succinogenes ◽

Gene Encoding ◽

Substrate Specificities ◽

Synergistic Interactions

ABSTRACT The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

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Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family.

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3417 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3417-3428 ◽

Cited By ~ 65

Author(s):

R T Nagao ◽

E Czarnecka ◽

W B Gurley ◽

F Schöffl ◽

J L Key

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Heat Shock ◽

Dna Sequences ◽

Regulatory Element ◽

Amino Acid Sequences ◽

Striking Similarity ◽

Low Molecular Weight ◽

Genes Encoding ◽

Flanking Regions

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.

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