Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4′)

Porter, Vanessa; Green, Keith D.; Zolova, Olga E.; Houghton, Jacob L.

doi:10.1016/j.bbrc.2010.10.119

Cited by 18 publications

(19 citation statements)

References 25 publications

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“…B. subtilis 168 was obtained from the Bacillus Genetic Stock Center (Columbus, OH). The C-terminally His tagged enzyme AAC(6Ј)/APH(2Љ)(CHis) (12), the N-terminally His tagged enzyme AAC(3)-IV(NHis) (from the Int-pET19b-pps vector) (12), ANT(4Ј)(NHis) (21), and APH(2Љ)(CHis) [amino acids 175 to 479 of the bifunctional AME AAC(6Ј)/APH(2Љ)] (22) were purified as described previously. The chemically competent bacteria Escherichia coli TOP10 and E. coli BL21(DE3) were obtained from Invitrogen (Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Alteration of enzymatic activities. In order to establish the effects of one modification on the second modification, all enzymes were cloned and purified as reported previously (2,12,15,21,22), except for AAC(3)-Ib/AAC(6Ј)-IbЈ, for which cloning into pET28a instead of pET22b resulted in the production of N-terminally His 6 tagged proteins instead of the previously reported untagged proteins (Fig. 1B).…”

Section: Antimicrobial Studiesmentioning

confidence: 99%

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Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

Green

Chen

2011

Antimicrob Agents Chemother

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Aminoglycoside-modifying enzymes (AMEs) constitute the most prevalent mechanism of resistance to aminoglycosides by bacteria. We show that aminoglycosides can be doubly modified by the sequential actions of AMEs, with the activity of the second AME in most cases unaffected, decreased, or completely abolished. We demonstrate that the bifunctional enzyme AAC(3)-Ib/AAC(6)-Ib can diacetylate gentamicin. Since single acetylation does not always inactivate the parent drugs completely, two modifications likely provide morerobust inactivation in vivo.

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Section: Methodsmentioning

confidence: 99%

Section: Antimicrobial Studiesmentioning

confidence: 99%

Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

Green

Chen

2011

Antimicrob Agents Chemother

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“…[13] We reasoned that the replacement of the 6”-primary alcohol of TOB with hydrophobic residues would interfere with these hydrophilic-based binding interactions and reduce the ability of the enzymes to modify these molecules. To test this hypothesis, the relative activities of seven different AMEs [14] on compounds 4a–r were compared to that of TOB (Figure 3). While some of the modified compounds served as better substrates for some AMEs, in general a drop in the AME catalytic activity was observed in most of the cases.…”

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confidence: 99%

6′′‐Thioether Tobramycin Analogues: Towards Selective Targeting of Bacterial Membranes

et al. 2012

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Amphiphilic tobramycin analogues with potent antibacterial activity against tobramycin‐resistant bacteria were synthesized. Most analogues were found to be less prone to deactivation by aminoglycoside‐modifying enzymes than tobramycin. These compounds target the bacterial membrane rather than the ribosome (see picture). The lipophilic residue of these analogues is key to their antibacterial potency and selectivity towards bacterial membranes.

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“…We previously demonstrated that AACs could display broad acyl-coenzyme A (CoA) cosubstrate promiscuity (20,21,34). Here, to gain insight into the mechanism of multiacetylation of Eis, we performed an in-depth study of the cosubstrate tolerance of Eis.…”

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confidence: 99%

Cosubstrate Tolerance of the Aminoglycoside Resistance Enzyme Eis from Mycobacterium tuberculosis

Chen

Green

2012

Antimicrob Agents Chemother

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We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6= position is the first to be npropionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.

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Dissecting the cosubstrate structure requirements of the Staphylococcus aureus aminoglycoside resistance enzyme ANT(4′)

Cited by 18 publications

References 25 publications

Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

6′′‐Thioether Tobramycin Analogues: Towards Selective Targeting of Bacterial Membranes

Cosubstrate Tolerance of the Aminoglycoside Resistance Enzyme Eis from Mycobacterium tuberculosis

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