Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

Mapping Intimacies ◽

10.1101/611251 ◽

2019 ◽

Author(s):

Murtala Muhammad ◽

Yangyang Li ◽

Siyu Gong ◽

Yanmin Shi ◽

Jiansong Ju ◽

...

Keyword(s):

Biochemical Characterization ◽

Antibiotic Activity ◽

Alanine Racemase ◽

Streptococcus Iniae ◽

Gram Negative Bacteria ◽

Reading Frame ◽

New Antibiotics ◽

Serious Disease ◽

Zoonotic Bacteria

ABSTRACTStreptococcus iniaeis a pathogenic and zoonotic bacteria that impacted high mortality to many fish species, as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5′-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from the pathogenic strain ofS. iniae, determined its biochemical characteristics and inhibitors. Thealrgene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The optimal enzyme activity occurred at 35°C and a pH of 9.5. The enzyme belongs to the PLP dependent enzymes family and is highly specific to L-alanine.S.iniaeAlr can be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parametersKmandVmaxof the enzyme were 33.11 mM, 2426 units/mg for L-alanine and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors, were determined and found to be effective against both gram positive and gram negative bacteria employed in this study. The important role of alanine racemase as a target of developing new antibiotics againstS. iniaehighlighted the usefulness of the enzyme for new antibiotics discovery.

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Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae

Polish Journal of Microbiology ◽

10.33073/pjm-2019-036 ◽

2019 ◽

Vol 68 (3) ◽

pp. 331-341 ◽

Cited By ~ 1

Author(s):

MURTALA MUHAMMAD ◽

YANGYANG LI ◽

SIYU GONG ◽

YANMIN SHI ◽

JIANSONG JU ◽

...

Keyword(s):

Homogentisic Acid ◽

Alanine Racemase ◽

Streptococcus Iniae ◽

Gram Negative Bacteria ◽

Chinese Sturgeon ◽

Reading Frame ◽

Optimal Activity ◽

Ic50 Values ◽

Serious Disease ◽

Zoonotic Bacteria

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S.iniaeAlr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters Km and Vmax of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

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Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure–function insights

Biochemical Journal ◽

10.1042/bj20060325 ◽

2006 ◽

Vol 398 (2) ◽

pp. 295-302 ◽

Cited By ~ 22

Author(s):

Cleyson Valença Reis ◽

Sonia Aparecida Andrade ◽

Oscar Henrique Pereira Ramos ◽

Celso Raul Romero Ramos ◽

Paulo Lee Ho ◽

...

Keyword(s):

Catalytic Activity ◽

Serine Protease ◽

Calcium Binding ◽

Biochemical Characterization ◽

Structural Features ◽

Reading Frame ◽

Molecular Features ◽

Recombinant Production ◽

Lonomia Obliqua

Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.

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Does a Ribosome Really Read? On the Cognitive Roots and Heuristic Value of Linguistic Metaphors in Molecular Genetics. Part 2

Russian Journal of Philosophical Sciences ◽

10.30727/0235-1188-2020-63-2-46-62 ◽

2020 ◽

Vol 63 (2) ◽

pp. 46-62

Author(s):

Suren T. Zolyan

Keyword(s):

Information Processing ◽

Genetic Information ◽

Formal Organization ◽

Dual Nature ◽

Reading Frame ◽

The Core ◽

Genetic Information Processing ◽

Cognitive Frame ◽

Effective Instrument

We discuss the role of linguistic metaphors as a cognitive frame for the understanding of genetic information processing. The essential similarity between language and genetic information processing has been recognized since the very beginning, and many prominent scholars have noted the possibility of considering genes and genomes as texts or languages. Most of the core terms in molecular biology are based on linguistic metaphors. The processing of genetic information is understood as some operations on text – writing, reading and editing and their specification (encoding/decoding, proofreading, transcription, translation, reading frame). The concept of gene reading can be traced from the archaic idea of the equation of Life and Nature with the Book. Thus, the genetics itself can be metaphorically represented as some operations on text (deciphering, understanding, code-breaking, transcribing, editing, etc.), which are performed by scientists. At the same time linguistic metaphors portrayed gene entities also as having the ability of reading. In the case of such “bio-reading” some essential features similar to the processes of human reading can be revealed: this is an ability to identify the biochemical sequences based on their function in an abstract system and distinguish between type and its contextual tokens of the same type. Metaphors seem to be an effective instrument for representation, as they make possible a two-dimensional description: biochemical by its experimental empirical results and textual based on the cognitive models of comprehension. In addition to their heuristic value, linguistic metaphors are based on the essential characteristics of genetic information derived from its dual nature: biochemical by its substance, textual (or quasi-textual) by its formal organization. It can be concluded that linguistic metaphors denoting biochemical objects and processes seem to be a method of description and explanation of these heterogeneous properties.

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MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5328

Author(s):

Miao Ma ◽

Margaux Lustig ◽

Michèle Salem ◽

Dominique Mengin-Lecreulx ◽

Gilles Phan ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Cell Division ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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Role of the Ser-287-Asn Replacement in the Hydrolysis Spectrum Extension of AmpC β-Lactamases in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00608-08 ◽

2008 ◽

Vol 53 (1) ◽

pp. 323-326 ◽

Cited By ~ 11

Author(s):

Hedi Mammeri ◽

Moreno Galleni ◽

Patrice Nordmann

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Catalytic Efficiency ◽

Phylogenetic Groups ◽

Extended Spectrum ◽

Group A

ABSTRACT Two AmpC variants harboring the S287N substitution were obtained by mutagenesis from cephalosporinases representative of the phylogenetic groups A and B2 of Escherichia coli. Their biochemical characterization revealed that the S287N replacement led to an important increase in the catalytic efficiency toward extended-spectrum cephalosporins in the AmpC β-lactamase of group A only.

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DNA methylation in Yersinia enterocolitica: role of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors

Microbiology ◽

10.1099/mic.0.27946-0 ◽

2005 ◽

Vol 151 (7) ◽

pp. 2291-2299 ◽

Cited By ~ 21

Author(s):

Stefan Fälker ◽

M. Alexander Schmidt ◽

Gerhard Heusipp

Keyword(s):

Mismatch Repair ◽

Yersinia Enterocolitica ◽

Virulence Genes ◽

Spontaneous Mutation ◽

Gram Negative Bacteria ◽

E Coli ◽

Physiological Processes ◽

Dna Adenine Methyltransferase

DNA adenine methyltransferase (Dam) plays an important role in physiological processes of Gram-negative bacteria such as mismatch repair and replication. In addition, Dam regulates the expression of virulence genes in various species. The authors cloned the dam gene of Yersinia enterocolitica and showed that Dam is essential for viability. Dam overproduction in Y. enterocolitica resulted in an increased frequency of spontaneous mutation and decreased resistance to 2-aminopurine; however, these effects were only marginal compared to the effect of overproduction of Escherichia coli-derived Dam in Y. enterocolitica, implying different roles or activities of Dam in mismatch repair of the two species. These differences in Dam function are not the cause for the essentiality of Dam in Y. enterocolitica, as Dam of E. coli can complement a dam defect in Y. enterocolitica. Instead, Dam seems to interfere with expression of essential genes. Furthermore, Dam mediates virulence of Y. enterocolitica. Dam overproduction results in increased tissue culture invasion of Y. enterocolitica, while the expression of specifically in vivo-expressed genes is not altered.

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One of the tightly clustered genes of the mouse surfeit locus is a highly expressed member of a multigene family whose other members are predominantly processed pseudogenes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3898 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3898-3905 ◽

Cited By ~ 21

Author(s):

C Huxley ◽

T Williams ◽

M Fried

Keyword(s):

Multigene Family ◽

Open Reading Frame ◽

The Other ◽

Base Pairs ◽

Regulation Of Expression ◽

Reading Frame ◽

Processed Pseudogenes ◽

Dna Fragment ◽

Basic Polypeptide

The mouse surfeit locus is unusual in that it contains a number of closely clustered genes (Surf-1, -2, and -4) that alternate in their direction of transcription (T. Williams, J. Yon, C. Huxley, and M. Fried, Proc. Natl. Acad. Sci. USA 85:3527-3530, 1988). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by 15 to 73 base pairs (bp), and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp (T. Williams and M. Fried, Mol. Cell. Biol. 6:4558-4569, 1986; T. Williams and M. Fried, Nature (London) 322:275-279, 1986). A fourth gene in this locus, Surf-3, which is a member of a multigene family, has been identified. The poly(A) addition site of Surf-3 lies only 70 bp from the poly(A) addition site of Surf-1. Transcription of Surf-3 has been studied in the absence of the other members of its multigene family after transfection of a cloned genomic mouse DNA fragment, containing the Surf-3 gene, into heterologous monkey cells. Surf-3 specifies a highly expressed 1.0-kilobase mRNA that contains a long open reading frame of 266 amino acids, which would encode a highly basic polypeptide (23% Arg plus Lys). The other members of the Surf-3 multigene family are predominantly, if not entirely, intronless pseudogenes with the hallmarks of being generated by reverse transcription. The role of the very tight clustering on regulation of expression of the genes in the surfeit locus is discussed.

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The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

Nature Communications ◽

10.1038/s41467-021-24432-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Paul White ◽

Samuel F. Haysom ◽

Matthew G. Iadanza ◽

Anna J. Higgins ◽

Jonathan M. Machin ◽

...

Keyword(s):

Outer Membrane Proteins ◽

Antibody Fragment ◽

Membrane Disruption ◽

Gram Negative Bacteria ◽

Wild Type ◽

Lipid Dynamics ◽

Open Structures

AbstractThe folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

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