Title
Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology
Publication Date
6-7-1994
Document Type
Article
Subjects
Aminoglycosides; Anti-Bacterial Agents; Base Sequence; Carbohydrate Sequence; Drug Resistance, Microbial; Enterococcus; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Kanamycin Kinase; Kinetics; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor); purification; Recombinant Proteins; Substrate Specificity
Disciplines
Biochemistry | Enzymes and Coenzymes | Medicinal-Pharmaceutical Chemistry | Therapeutics
Abstract
The aminoglycoside phosphotransferases (APHs) are responsible for the bacterial inactivation of many clinically useful aminoglycoside antibiotics. We report the characterization of an enterococcal enzyme, APH(3')-IIIa, which inactivates a broad spectrum of aminoglycosides by ATP-dependent O-phosphorylation. Overproduction of APH(3')-IIIa has permitted the isolation of 30-40 mg of pure protein/(L of cell culture). Purified APH(3')-IIIa is a mixture of monomer and dimer which is slowly converted to dimer only over time. Dimer could be dissociated into monomer by incubation with 2-mercaptoethanol, suggesting that dimerization is mediated by formation of disulfide bond(s). Both monomer and dimer show Km values in the low micromolar range for good substrates such as kanamycin and neomycin, and kcat values of 1-4 s-1. All aminoglycosides show substrate inhibition except amikacin and kanamycin B. Determination of minimum inhibitory concentrations indicates a positive correlation between antibiotic activity and kcat/Km, but not with Km or kcat. NMR analysis of phosphorylated kanamycin A has directly demonstrated regiospecific phosphoryl transfer to the 3'-hydroxyl of the 6-aminohexose ring of the antibiotic. Analysis of structure-activity relationships with a variety of aminoglycosides has revealed that the deoxystreptamine aminocyclitol ring plays a critical role in substrate binding. This information will form the basis for future design of inhibitors of APH(3')-IIIa.
DOI of Published Version
10.1021/bi00188a024
Source
Biochemistry. 1994 Jun 7;33(22):6936-44. doi:10.1021/bi00188a024
Journal/Book/Conference Title
Biochemistry
Related Resources
Repository Citation
McKay, G. A.; Thompson, Paul R.; and Wright, G. D., "Broad spectrum aminoglycoside phosphotransferase type III from Enterococcus: overexpression, purification, and substrate specificity" (1994). Thompson Lab Publications. 99.
https://escholarship.umassmed.edu/thompson/99
Comments
At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.