Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

Keith D. Green; Sylvie Garneau-Tsodikova

doi:10.1016/j.biochi.2013.02.011

Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

Keith D. Green
, Sylvie Garneau-Tsodikova

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3″)-Ii/AAC(6′)-IId. To gain insight into the role, effect, and importance of the union of ANT(3″)-Ii and AAC(6′)-IId in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3″)-Ii/AAC(6′)-IId and its individually expressed components.

Original language	English
Pages (from-to)	1319-1325
Number of pages	7
Journal	Biochimie
Volume	95
Issue number	6
DOIs	https://doi.org/10.1016/j.biochi.2013.02.011
State	Published - Jun 2013

Bibliographical note

Funding Information:
This work was supported by a grant from the Firland Foundation (S.G.-T.), a BSF grant 2008017 (S.G.-T.), and a National Institute of Health (NIH) grant AI090048 (S.G.-T.) We thank Vanessa Porter for cloning and preliminary experimental work. We thank Dr. Paul H. Roy for the Serratia marcescens genomic DNA and Dr. Tapan Biswas for the Int-pET19b-pps vector. We thank Oleg V. Tsodikov for critical reading and insightful comments on the manuscript.

Funding

This work was supported by a grant from the Firland Foundation (S.G.-T.), a BSF grant 2008017 (S.G.-T.), and a National Institute of Health (NIH) grant AI090048 (S.G.-T.) We thank Vanessa Porter for cloning and preliminary experimental work. We thank Dr. Paul H. Roy for the Serratia marcescens genomic DNA and Dr. Tapan Biswas for the Int-pET19b-pps vector. We thank Oleg V. Tsodikov for critical reading and insightful comments on the manuscript.

Funders	Funder number
National Institutes of Health (NIH)
National Institute of Allergy and Infectious Diseases	R01AI090048
Firland Foundation

Keywords

Acetyltransferase
Aminoglycoside antibiotics
Bacterial resistance
Bifunctional enzyme
Nucleotidyltransferase

ASJC Scopus subject areas

Biochemistry

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10.1016/j.biochi.2013.02.011

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title = "Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens",

abstract = "Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3″)-Ii/AAC(6′)-IId. To gain insight into the role, effect, and importance of the union of ANT(3″)-Ii and AAC(6′)-IId in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3″)-Ii/AAC(6′)-IId and its individually expressed components.",

keywords = "Acetyltransferase, Aminoglycoside antibiotics, Bacterial resistance, Bifunctional enzyme, Nucleotidyltransferase",

author = "Green, \{Keith D.\} and Sylvie Garneau-Tsodikova",

note = "Funding Information: This work was supported by a grant from the Firland Foundation (S.G.-T.), a BSF grant 2008017 (S.G.-T.), and a National Institute of Health (NIH) grant AI090048 (S.G.-T.) We thank Vanessa Porter for cloning and preliminary experimental work. We thank Dr. Paul H. Roy for the Serratia marcescens genomic DNA and Dr. Tapan Biswas for the Int-pET19b-pps vector. We thank Oleg V. Tsodikov for critical reading and insightful comments on the manuscript. ",

year = "2013",

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language = "English",

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N1 - Funding Information: This work was supported by a grant from the Firland Foundation (S.G.-T.), a BSF grant 2008017 (S.G.-T.), and a National Institute of Health (NIH) grant AI090048 (S.G.-T.) We thank Vanessa Porter for cloning and preliminary experimental work. We thank Dr. Paul H. Roy for the Serratia marcescens genomic DNA and Dr. Tapan Biswas for the Int-pET19b-pps vector. We thank Oleg V. Tsodikov for critical reading and insightful comments on the manuscript.

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N2 - Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3″)-Ii/AAC(6′)-IId. To gain insight into the role, effect, and importance of the union of ANT(3″)-Ii and AAC(6′)-IId in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3″)-Ii/AAC(6′)-IId and its individually expressed components.

AB - Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3″)-Ii/AAC(6′)-IId. To gain insight into the role, effect, and importance of the union of ANT(3″)-Ii and AAC(6′)-IId in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3″)-Ii/AAC(6′)-IId and its individually expressed components.

KW - Acetyltransferase

KW - Aminoglycoside antibiotics

KW - Bacterial resistance

KW - Bifunctional enzyme

KW - Nucleotidyltransferase

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Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

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