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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Jun 1;67(Pt 6):703-6. doi: 10.1107/S1744309111013832. Epub 2011 May 26.

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

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1
Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, Belgium.

Abstract

TEM-1 β-lactamase is a highly efficient enzyme that is involved in bacterial resistance against β-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 β-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native β-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.

PMID:
21636917
PMCID:
PMC3107148
DOI:
10.1107/S1744309111013832
[Indexed for MEDLINE]
Free PMC Article

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