Biochemical and structural analysis of aminoglycoside acetyltransferase Eis from Anabaena variabilis

Pricer, Rachel; Houghton, Jacob L.; Green, Keith D.; Mayhoub, Abdelrahman S.

doi:10.1039/c2mb25341k

Cited by 31 publications

(63 citation statements)

References 17 publications

(44 reference statements)

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“…8,9 We have also performed studies monitoring the effect that different modifications have on a second modification and its catalyzing enzyme. 9 We found that Eis from Mtb (Eis_ Mtb ) is capable of multiacetylating AGs, 2 and we observed a similar multiacetylating behavior when studying Eis from Mycobacterium smegmatis (Eis_ Msm 1), 10 Eis from Anabaena variabilis (Eis_ Ava ), 11 and the Eis homologue from Bacillus anthracis (Eis_ Ban ). 37 AACs are known to be cosubstrate promiscuous, 8 although we have shown that Eis_ Mtb has a much more limited cosubstrate tolerance.…”

supporting

confidence: 61%

“…2,3,10,11,33,37 Shown in Figure 4 are the four currently known crystal structures of Eis_ Mtb and its three crystallized homologues. The cartoon overlay of the four crystal structures (Figure 4A) shows only minor changes in the backbone of the structure with only one or two misaligned α -helices and a few disordered loops.…”

Section: Resultsmentioning

confidence: 99%

“…Liquid chromatography–mass spectrometry (LC-MS) chromatograms were obtained on a Shimazdu LC-MS-2019EV equipped with an SPD-20AV UV–vis detector and an LC-20AD liquid chromatograph. The PDB structures 2OZG (Eis_ Ava ), 11 3N7Z (Eis_ Ban ), 37 3SXN (Eis_ Msm 1), 17 and 3R1K (Eis_ Mtb ) 2 were visualized using PyMOL (The PyMOL Molecular Graphics System, version 1.4.1, Schrödinger, LLC).…”

Section: Methodsmentioning

confidence: 99%

“…Eis_ Mtb , 2 Eis_ Msm 1, 10 Eis_ Ban , 37 and Eis_ Ava 11 were cloned and purified as previously described. The remaining Eis proteins were cloned using the isolated DNA, the corresponding primers reported in Table S2, and Phusion DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

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Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria

Green

Pricer²,

Stewart³

2015

ACS Infect. Dis.

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Antibiotic resistance is a growing problem worldwide. Of particular importance is the resistance of Mycobacterium tuberculosis (Mtb) to currently available antibiotics used in the treatment of infected patients. Up-regulation of an aminoglycoside (AG) acetyltransferase, the enhanced intracellular survival (Eis) protein of Mtb (Eis_Mtb), is responsible for resistance to the second-line injectable drug kanamycin A in a number of Mtb clinical isolates. This acetyltransferase is known to modify AGs, not at a single position, as usual for this type of enzyme, but at multiple amine sites. We identified, using in silico techniques, 22 homologues from a wide variety of bacteria, that we then cloned, purified, and biochemically studied. From the selected Eis homologues, 7 showed the ability to modify AGs to various degrees and displayed both similarities and differences when compared to Eis_Mtb. In addition, an inhibitor proved to be active against all homologues tested. Our findings show that this family of acetyltransferase enzymes exists in both mycobacteria and non-mycobacteria and in both pathogenic and nonpathogenic species. The bacterial strains described herein should be monitored for rising resistance rates to AGs.

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supporting

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria

Green

Pricer²,

Stewart³

2015

ACS Infect. Dis.

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“…There are three types of AMEs: the aminoglycoside N-acetyltransferases (AACs), the aminoglycoside O-phosphotransferases (APHs), and the aminoglycoside O-nucleotidyltransferases (ANTs). Recently, a unique AAC, the enhanced intracellular survival (Eis) protein from a variety of bacterial species, was found capable of multiacetylating aminoglycosides (12,(28)(29)(30)(31)(32)(33)(34)(35)(36). Knowing that our compounds had good antibacterial activity, we tested the novel C 4 compound to see if it became modified by AMEs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents

Shrestha

Fosso

Green

et al. 2015

Antimicrob Agents Chemother

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In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studies. Cytotoxicity was evaluated in mammalian cell lines. The fungal membrane-disruptive action of these analogues was studied by using the membrane-impermeable dye propidium iodide. TOB analogues bearing a linear alkyl chain at their 6؆-position in a thioether linkage exhibited chain length-dependent antifungal activities. Analogues with C 12 and C 14 chains showed promising antifungal activities against tested fungal strains, with MIC values ranging from 1.95 to 62.5 mg/liter and 1.95 to 7.8 mg/liter, respectively. However, C 4 , C 6 , and C 8 TOB analogues and TOB itself exhibited little to no antifungal activity. Fifty percent inhibitory concentrations (IC 50 s) for the most potent TOB analogues (C 12 and C 14 ) against A549 and Beas 2B cells were 4-to 64-fold and 32-to 64-fold higher, respectively, than their antifungal MIC values against various fungi. Unlike conventional aminoglycoside antibiotics, TOB analogues with alkyl chain lengths of C 12 and C 14 appear to inhibit fungi by inducing apoptosis and disrupting the fungal membrane as a novel mechanism of action. Amphiphilic TOB analogues showed broad-spectrum antifungal activities with minimal mammalian cell cytotoxicity. This study provides novel lead compounds for the development of antifungal drugs.

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Chemical and Structural Insights into the Regioversatility of the Aminoglycoside Acetyltransferase Eis

et al. 2013

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A recently discovered cause of resistance of tuberculosis to a drug of last resort, the aminoglycoside kanamycin, is modification of this drug by the enhanced intracellular survival (Eis) protein. Eis is a structurally and functionally unique acetyltransferase with an unusual capability of acetylating aminoglycosides at multiple positions. The extent of this regioversatility and its defining protein features are unclear. Herein, we determined the positions and order of acetylation of five aminoglycosides by NMR spectroscopy. This analysis revealed unprecedented acetylation of the 3"-amine of kanamycin, amikacin, and tobramycin, and γ-amine of the 4-amino-2-hydroxybutyryl group of amikacin. A crystal structure of Eis in complex with coenzyme A and tobramycin revealed how tobramycin can be accommodated in the Eis active site in two binding modes consistent with its di-acetylation. These studies describing chemical and structural details of acetylation will guide future efforts towards designing aminoglycosides and Eis inhibitors to overcome resistance in tuberculosis.

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Biochemical and structural analysis of aminoglycoside acetyltransferase Eis from Anabaena variabilis

Cited by 31 publications

References 17 publications

Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria

Comparative Study of Eis-like Enzymes from Pathogenic and Nonpathogenic Bacteria

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents

Chemical and Structural Insights into the Regioversatility of the Aminoglycoside Acetyltransferase Eis

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