Papadovasilaki Maria, Oberthür Dominik, Gessmann Renate, Sarrou Iosifina, Betzel Christian, Scoulica Effie, Petratos Kyriacos
Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology-Hellas, N. Plastira 100, Heraklion 70013, Greece.
Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University Hamburg, Martin-Luther-King Platz 6, Hamburg 20146, Germany.
Biochem Biophys Rep. 2015 Sep 18;4:152-157. doi: 10.1016/j.bbrep.2015.09.011. eCollection 2015 Dec.
The gene coding for the aminoglycoside adenylyltransferase () from a clinical isolate of was cloned and expressed in strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl for normal Michaelis-Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.
从临床分离株中编码氨基糖苷腺苷酸转移酶（）的基因被克隆，并在大肠杆菌BL21(DE3)pLysS菌株中表达。将过表达的酶（AadA6，281个氨基酸残基）和一个羧基末端截短的变体分子（[1-264]AadA6）纯化至接近均一，并进行了表征。在低离子强度下进行的光散射实验支持酶亚基的单体和同型二聚体排列之间的平衡。圆二色光谱偏振法表明其与腺苷酸激酶有密切的结构关系。两种形式均共价修饰氨基糖苷类的链霉素和壮观霉素。该酶正常的米氏动力学至少需要5 mM MgCl₂。在1 mM MgCl₂时链霉素表现出强烈的底物抑制作用。C末端截短的17个残基对蛋白质折叠影响很小，而它们对酶活性有积极作用，并在高蛋白浓度（>100 μM）下稳定二聚体。基于已知晶体结构的同源建模和对接产生了单体AadA6与ATP以及链霉素或壮观霉素的中心三元复合物模型。
