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DOE OSTI2016

The structure of a doripenem‐bound OXA‐51 class D β‐lactamase variant with enhanced carbapenemase activity

Abstract OXA‐51 is a class D β‐lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii . Many variants of OXA‐51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high‐resolution structures of apo OXA‐51 and OXA‐51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl‐intermediate. The structure shows that acyl‐doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA‐24/40 (doripenem) and OXA‐23 (meropenem). In the OXA‐51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA‐51. Similarly, in the OXA‐51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA‐51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA‐51 and OXA‐51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA‐51 variants.

June, Cynthia M.↗
DOE OSTI2016

Glutathione transferase P1‐1 as an arsenic drug‐sequestering enzyme

Abstract Arsenic‐based compounds are paradoxically both poisons and drugs. Glutathione transferase (GSTP1‐1) is a major factor in resistance to such drugs. Here we describe using crystallography, X‐ray absorption spectroscopy, mutagenesis, mass spectrometry, and kinetic studies how GSTP1‐1 recognizes the drug phenylarsine oxide (PAO). In conditions of cellular stress where glutathione (GSH) levels are low, PAO crosslinks C47 to C101 of the opposing monomer, a distance of 19.9 Å, and causes a dramatic widening of the dimer interface by approximately 10 Å. The GSH conjugate of PAO, which forms rapidly in cancerous cells, is a potent inhibitor ( K i = 90 n M ) and binds as a di‐GSH complex in the active site forming part of a continuous network of interactions from one active site to the other. In summary, GSTP1‐1 can detoxify arsenic‐based drugs by sequestration at the active site and at the dimer interface, in situations where there is a plentiful supply of GSH, and at the reactive cysteines in conditions of low GSH.

Parker, Lorien J.↗
DOE OSTI2026

Identification of a key water molecule involved in the macrophage migration inhibitory factor‐catalyzed tautomerization of para ‐hydroxyphenylpyruvate using neutron crystallography

Abstract Neutron crystallography was used to determine a 2.5‐Å resolution all‐atom structure of macrophage migration inhibitory factor (MIF) interacting with 3‐(4‐hydroxyphenyl)‐pyruvate (HPP). MIF is a pro‐inflammatory, pro‐tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF‐catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction‐derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine‐32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N‐terminal Pro‐1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room‐temperature synchrotron x‐ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation‐induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.

Schröder, Gabriela C. [Department of Molecular and↗