Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis

Abstract

The emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of βlactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and pK a s of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with Nζ of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 is activated for nucleophilic attack of the β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the acyl-enzyme intermediate.

Probing the role of the conserved residue Glu166 in a class A β-lactamase using neutron and X-ray protein crystallography

“…Following the formation of the acylation tetrahedral intermediate, protonation of the -lactam N atom must occur to break the -lactam ring and form the acyl-enzyme adduct. In recent studies (Meroueh et al, 2005;Vandavasi et al, 2016Vandavasi et al, , 2017Langan et al, 2018), it has been proposed that the -lactam ring is opened by the transfer of a proton from the NZ group of Lys73 to the OG group of Ser130 followed by the simultaneous transfer of a proton from the Ser130 OG group to the -lactam ring N atom (Meroueh et al, 2005;. The final step in the acylation pathway involves proton transfer from Lys73 to Glu166, which initiates the start of the deacylation reaction.…”

Neutron scattering in the biological sciences: progress and prospects

“…Example (2015)(2016)(2017)(2018) Protonation states to elucidate enzymatic mechanism Xylose isomerase (Meilleur, Snell et al, 2006), chlorite dismutase (Schaffner et al, 2017), T4 lysozyme (Hiromoto et al, 2017), phosphoactive yellow protein (Yonezawa et al, 2017), RAS (Knihtila et al, 2015), Cel45A (Nakamura et al, 2015), phycocyanobilin:ferredoxin oxidoreductase (Unno et al, 2015) Protein-ligand interaction/protein-drug complex Galectin 3C (Manzoni et al, 2018), xylose isomerase (Munshi et al, 2014), PKG (Gerlits et al, 2018), -lactamase (Langan et al, 2018), trypsin (Schiebel et al, 2017), GCN5-related N-acetyltransferase (Kumar et al, 2018), pyridoxal 5 0 -phosphate enzyme (Dajnowicz et al, 2017), concanavalin A (Gerlits, Coates et al, 2017), MTAN (Banco et al, 2016), farnesyl pyrophosphate synthase (Yokoyama et al, 2015) Titration (pH studies) HIV (Gerlits et al, 2016), RAS (Knihtila, 2016), xylanase (Wan et al, 2015) Hydration and water binding at the active site of enzymes Carbonic anhydrase (Kovalevsky et al, 2018), RAS (Knihtila et al, 2015), H-FABP (Howard et al, 2016), carbohydrate-binding module (Fisher et al, 2015), hydronium ion identification (Kovalevsky et al, 2011) Metalloprotein/redox chemistry Lytic polysaccharide monooxygenase (Bacik et al, 2017;O'Dell et al, 2017), cholesterol oxidase (Golden et al, 2017), ascorbate peroxidase (Kwon et al, 2016), cytochrome c peroxidase (Kwon et al, 2016) Room ('physiological') temperature structures † HIV (Gerlits, Keen et al, 2017) † Most neutron crystallographic data are collected at room temperature. Here, we list proteins for which neutron diffraction at room temperature revealed st...…”

The rise of neutron cryo-crystallography

“…They are the leading cause of resistance to this class of potent and widely used antibiotics and as such are a major health problem, increasing surgical complications and mortality rates (Livermore & Woodford, 2006). Several neutron structures of Toho1 -lactamase have been published that have studied the role of the catalytic residue Glu166 in the acylation reaction (Tomanicek et al, 2013), while later studies probed the role of the catalytic residue Lys73 in the opening of the -lactam ring (Vandavasi et al, 2016(Vandavasi et al, , 2017 and the changes that occur upon substrate binding (Langan et al, 2018). However, even engineered mutants of -lactamase enzymes in which the catalytic residues are mutated still show activity against several types of antibiotics and -lactamase inhibitors (Nitanai et al, 2010;King et al, 2015).…”