5CFU

Crystal Structure of ANT(2")-Ia in complex with adenylyl-2"-tobramycin

5CFU の概要

• エントリーDOI: 10.2210/pdb5cfu/pdb
• 関連するPDBエントリー: 4XJE 5CFS 5CFT
• 分子名称: Aminoglycoside Nucleotidyltransferase (2")-Ia, MANGANESE (II) ION, adenylyl-2"-tobramycin, ... (6 entities in total)
• 機能のキーワード: antibiotic resistance, nucleotidyltransferase, ampcpp, tobramycin, modified aminoglycoside, rossmann fold, transferase-antibiotic complex, transferase/antibiotic
• 由来する生物種: Pseudomonas aeruginosa
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 22493.40

構造登録者

Rodionov, D.,Bassenden, A.V.,Berghuis, A.M. (登録日: 2015-07-08, 公開日: 2016-07-20, 最終更新日: 2023-09-27)

主引用文献

Bassenden, A.V.,Park, J.,Rodionov, D.,Berghuis, A.M.
Revisiting the Catalytic Cycle and Kinetic Mechanism of AminoglycosideO-Nucleotidyltransferase(2′′): A Structural and Kinetic Study.
Acs Chem.Biol., 2020

PubMed Abstract: Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is aminoglycoside -nucleotidyltransferase(2″), which catalyzes a nucleotidylation reaction. Due to its clinical importance, much research has focused on dissecting the mechanism of action, some of it dating back more than 30 years. Here, we present structural data for catalytically informative states of the enzyme, i.e., ANT(2″) in complex with adenosine monophosphate (AMP) and tobramycin (inactive-intermediate state) and in complex with adenylyl-2″-tobramycin, pyrophosphate, and Mn(product-bound state). These two structures in conjunction with our previously reported structure of ANT(2″)'s substrate-bound complex capture clinical states along ANT(2″)'s reaction coordinate. Additionally, isothermal titration calorimetry (ITC)-based studies are presented that assess the order of substrate binding and product release. Combined, these results outline a kinetic mechanism for ANT(2″) that contradicts what has been previously reported. Specifically, we show that the release of adenylated aminoglycoside precedes pyrophosphate. Furthermore, the ternary complex structures provide additional details on the catalytic mechanism, which reveals extensive similarities to the evolutionarily related DNA polymerase-β superfamily.

PubMed: 32100995
DOI: 10.1021/acschembio.9b00904

実験手法

X-RAY DIFFRACTION (1.82 Å)