Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding ?-lactamase in complex with its ligand.

ABSTRACT: TEM-1 ?-lactamase is a highly efficient enzyme that is involved in bacterial resistance against ?-lactam antibiotics such as penicillin. It is also a robust scaffold protein which can be engineered by molecular-evolution techniques to bind a variety of targets. One such ?-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native ?-lactamases. In addition to recognizing kan, BlaKr activity is up-regulated by its binding via an activation mechanism which is not yet understood at the molecular level. In order to fill this gap, determination of the structure of the BlaKr-kan complex was embarked upon. A crystallization condition for BlaKr-kan was identified using high-throughput screening, and crystal growth was further optimized using streak-seeding and hanging-drop methods. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.01, b = 72.33, c = 74.62?Å, and diffracted to 1.67?Å resolution using synchrotron radiation. The X-ray structure of BlaKr with its ligand kanamycin should provide the molecular-level details necessary for understanding the activation mechanism of the engineered enzyme.

SUBMITTER: Van de Water K 

PROVIDER: S-EPMC3107148 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature