Plasmid pRARE as a Vector for Cloning to Construct a Superproducer of the Site-Specific Nickase N.BspD6I

Rogulin, E. A.; Perevyazova, T. A.; Zheleznaya, L. A.; Matvienko, N. I.

doi:10.1023/b:biry.0000046886.19428.d5

Cited by 20 publications

(8 citation statements)

References 9 publications

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“…The constructed plasmids were transformed into in-house-modified E. coli BL21 Star(DE3)pLysS (Invitrogen) cells containing the pRARE plasmid (Novagen), allowing expression of genes encoding tRNAs for rare codons (21). The precultures were grown overnight in LB containing 100 g/ml ampicillin (Sigma-Aldrich) and 34 g/ml chloramphenicol (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Skagseth

Carlsen

Bjerga

et al. 2016

Antimicrob Agents Chemother

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e Metallo-␤-lactamases (MBLs) hydrolyze virtually all ␤-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ␤-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ␤-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ␤-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1. Members of the carbapenem class of ␤-lactams are among the most important antimicrobial agents available for the treatment of serious bacterial infections (1). However, their use against Gram-negative bacteria is now threatened by the dissemination of carbapenemases, ␤-lactamases that hydrolyze the amide bond of the ␤-lactam ring, thereby inactivating carbapenems and other ␤-lactams (2). To date, only a few effective carbapenemase inhibitors have been available for clinical use. Enzymes with carbapenemase activity have been identified in multiple ␤-lactamase classes. ␤-Lactamases are divided into four classes, of which classes A, C, and D are serine enzymes that catalyze hydrolysis of the ␤-lactam through a serine-bound acyl intermediate, whereas class B ␤-lactamases, or metallo-␤-lactamases (MBLs), coordinate one or two zinc ions in the active site, which are essential for their enzymatic activity (1-3). The class B MBLs can be divided into four subclasses, according to their primary structure: B1a (e.g., VIM, IMP, DIM, and SPM), B1b (NDM), B2 (e.g., CphA), and B3 (e.g., L1 and AIM) (3). Currently, no clinically effective MBL inhibitor has been approved for use (4). Avibactam, the only clinically approved effective carbap...

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Section: Methodsmentioning

confidence: 99%

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Skagseth

Carlsen

Bjerga

et al. 2016

Antimicrob Agents Chemother

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“…N-terminal tagging of MamK and MamK-like with the SUMO tag allows affinity purification of the proteins and seamless removal of the tag by the action of the SUMO protease. The pET-SUMO plasmids were cotransformed into One Shot BL21 Star (DE3) chemically competent Escherichia coli cells along with the pRARE plasmid carrying several rare tRNAs (36). Transformants were selected on LB medium in the presence of both kanamycin (50 g/ml) and chloramphenicol (25 g/ml).…”

Section: Methodsmentioning

confidence: 99%

Interplay between Two Bacterial Actin Homologs, MamK and MamK-Like, Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

et al. 2014

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dMany bacterial species contain multiple actin-like proteins tasked with the execution of crucial cell biological functions. MamK, an actin-like protein found in magnetotactic bacteria, is important in organizing magnetosome organelles into chains that are used for navigation along geomagnetic fields. MamK and numerous other magnetosome formation factors are encoded by a genetic island termed the magnetosome island. Unlike most magnetotactic bacteria, Magnetospirillum magneticum AMB-1 (AMB-1) contains a second island of magnetosome-related genes that was named the magnetosome islet. A homologous copy of mamK, mamK-like, resides within this islet and encodes a protein capable of filament formation in vitro. Previous work had shown that mamK-like is expressed in vivo, but its function, if any, had remained unknown. Though MamK-like is highly similar to MamK, it contains a mutation that in MamK and other actins blocks ATPase activity in vitro and filament dynamics in vivo. Here, using genetic analysis, we demonstrate that mamK-like has an in vivo role in assisting organelle alignment. In addition, MamK-like forms filaments in vivo in a manner that is dependent on the presence of MamK and the two proteins interact in a yeast two-hybrid assay. Surprisingly, despite the ATPase active-site mutation, MamK-like is capable of ATP hydrolysis in vitro and promotes MamK filament turnover in vivo. Taken together, these experiments suggest that direct interactions between MamK and MamK-like contribute to magnetosome alignment in AMB-1.

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“…Crystallization and data collection N.BspD6I, 48 which has recently been renamed Nt. BspD6I, and ss.BspD6I 15 were expressed in Escherichia coli.…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Heterodimeric Type IIS Restriction Endonuclease R.BspD6I Acting as a Complex between a Monomeric Site-specific Nickase and a Catalytic Subunit

Качалова

Rogulin

Yunusova

et al. 2008

Journal of Molecular Biology

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Plasmid pRARE as a Vector for Cloning to Construct a Superproducer of the Site-Specific Nickase N.BspD6I

Cited by 20 publications

References 9 publications

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Interplay between Two Bacterial Actin Homologs, MamK and MamK-Like, Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

Structural Analysis of the Heterodimeric Type IIS Restriction Endonuclease R.BspD6I Acting as a Complex between a Monomeric Site-specific Nickase and a Catalytic Subunit

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