4BD0

X-ray structure of a perdeuterated Toho-1 R274N R276N double mutant Beta-lactamase in complex with a fully deuterated boronic acid (BZB)

Summary for 4BD0

• Entry DOI: 10.2210/pdb4bd0/pdb
• Related: 1BZA 1IYO 1IYP 1IYQ 1IYS 1WE4 2WYX 2XQZ 2XR0 4BD1
• Descriptor: BETA-LACTAMASE TOHO-1, BENZO[B]THIOPHENE-2-BORONIC ACID, SULFATE ION, ... (4 entities in total)
• Functional Keywords: hydrolase, perdeuterated neutron structure, extended-spectrum beta lactamases, ctx- m-type esbls
• Biological source: ESCHERICHIA COLI BL21
• Total number of polymer chains: 1
• Total formula weight: 28859.11

Authors

Tomanicek, S.J.,Weiss, K.L.,Standaert, R.F.,Ostermann, A.,Schrader, T.E.,Ng, J.D.,Coates, L. (deposition date: 2012-10-04, release date: 2013-01-09, Last modification date: 2023-12-20)

Primary citation

Tomanicek, S.J.,Standaert, R.F.,Weiss, K.L.,Ostermann, A.,Schrader, T.E.,Ng, J.D.,Coates, L.
Neutron and X-Ray Crystal Structures of a Perdeuterated Enzyme Inhibitor Complex Reveal the Catalytic Proton Network of the Toho-1 Beta-Lactamase for the Acylation Reaction.
J.Biol.Chem., 288:4715-, 2013

PubMed Abstract: The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with (11)B, as (10)B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction.

PubMed: 23255594
DOI: 10.1074/JBC.M112.436238

Experimental method

X-RAY DIFFRACTION (1.207 Å)