Aminoglycoside 2″-Phosphotransferase Type IIIa from Enterococcus

Adriana Badarau,Qicun Shi,Joseph W Chow,Jaroslav Zajicek,Shahriar Mobashery,Sergei Vakulenko

doi:10.1074/jbc.m709645200

Abstract

Aminoglycoside 2''-phosphotransferases mediate high level resistance to aminoglycoside antibiotics in Gram-positive microorganisms, thus posing a serious threat to the treatment of serious enterococcal infections. This work reports on cloning, purification, and detailed mechanistic characterization of aminoglycoside 2''-phosphotransferase, known as type Ic enzyme. In an unexpected finding, the enzyme exhibits strong preference for guanosine triphosphate over adenosine triphosphate as the phosphate donor, a unique observation among all characterized aminoglycoside phosphotransferases. The enzyme phosphorylates only certain 4,6-disubstituted aminoglycosides exclusively at the 2''-hydroxyl with k(cat) values of 0.5-1.0 s(-1) and K(m) values in the nanomolar range for all substrates but kanamycin A. Based on this unique substrate profile, the enzyme is renamed aminoglycoside 2''-phosphotransferase type IIIa. Product and dead-end inhibition patterns indicated a random sequential Bi Bi mechanism. Both the solvent viscosity effect and determination of the rate constant for dissociation of guanosine triphosphate indicated that at pH 7.5 the release of guanosine triphosphate is rate-limiting. A computational model for the enzyme is presented that sheds light on the structural aspects of interest in this family of enzymes.

Highlights

The main mechanism of resistance of bacteria to aminoglycoside antibiotics is the enzymatic modification of the amino or hydroxyl groups of these drugs [1]
The kcat values are similar for GTP, ITP, UTP, and ATP with kanamycin B, dibekacin, and tobramycin, there was a larger difference in catalytic efficiency of the enzyme for kanamycin A when different NTPs were used as phosphate donor (Table 1)
The enzyme designated as Aminoglycoside phosphotransferases (APHs)(2Љ)-IIIa here belongs to a class of proteins that confers bacterial resistance to aminoglycosides by the phosphorylation of the 2Љ-position of these antibiotics

Summary

Introduction

The main mechanism of resistance of bacteria to aminoglycoside antibiotics is the enzymatic modification of the amino or hydroxyl groups of these drugs [1]. The presence of the 2Ј amino group increases the turnover numbers and lowers aminoglycoside Km values for APH(2Љ)-IIIa with ATP and UTP as second substrates (Table 1).

Results

Conclusion