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Due to their highly strained structures, epoxides can readily undergo ring-opening reactions through nucleophilic substitution, either in the presence of an acid or a base. The nucleophilic substitution reactions in the presence of acid are called acid-catalyzed ring-opening reactions, and nucleophilic substitution reactions in the presence of a base are called base-catalyzed ring-opening reactions. Epoxides undergo base-catalyzed ring-opening reactions in the presence of a strong nucleophile...
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Historical perspective
In 1896, the German chemist Paul Walden discovered that he could interconvert pure enantiomeric (+) and (-) malic acids through a series of reactions. This conversion suggested the involvement of optical inversion during the substitution reaction. Further, in 1930, Sir Christopher Ingold described for the first time two different forms of nucleophilic substitution reactions, which are known as SN1 (nucleophilic substitution unimolecular) and SN2 (nucleophilic substitution...
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Baeyer–Villiger oxidation converts aldehydes to carboxylic acids and ketones to esters. The reaction uses peroxy acids or peracids and is often catalyzed by acid. The reaction is named after its pioneers, Adolf von Baeyer and Victor Villiger. The reaction is achieved by a wide range of peracids such as m-chloroperoxybenzoic acid (mCPBA), perbenzoic acid (C6H5COOOH), peracetic acid (CH3COOOH), hydrogen peroxide (H2O2), and tert-butyl hydroperoxide (t-BuOOH).
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In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.
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A significant aspect of hydroboration–oxidation is the regio- and stereochemical outcome of the reaction.
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Substrate Specificity of OXA-48 after β5-β6 Loop Replacement.

Laura Dabos1,2, Agustin Zavala3, Rémy A Bonnin1,2,4

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ACS Infectious Diseases
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Summary

Researchers modified the OXA-48 carbapenemase enzyme by altering its β5-β6 loop. This change expanded its ability to hydrolyze expanded-spectrum cephalosporins while retaining carbapenemase activity, revealing enzyme plasticity.

Keywords:
carbapenemaseexpanded-spectrum cephalosporinsoxacillinasesβ5−β6 loop

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Area of Science:

  • Microbiology
  • Biochemistry
  • Structural Biology

Background:

  • OXA-48 carbapenemase is a significant global health threat due to its rapid spread.
  • Variants of OXA-48 carbapenemase exhibit altered substrate hydrolysis profiles, often linked to modifications in the β5-β6 loop.
  • Deletions in the β5-β6 loop can lead to loss of carbapenemase activity and gain of expanded-spectrum cephalosporin (ESC) hydrolysis.

Purpose of the Study:

  • To investigate the role of the β5-β6 loop in the substrate selectivity of OXA-48-like enzymes.
  • To engineer a hybrid enzyme with altered hydrolytic capabilities by replacing the OXA-48 β5-β6 loop with that of OXA-18.
  • To elucidate the structure-function relationship of β-lactamases concerning loop conformation and substrate binding.

Main Methods:

  • Enzyme engineering: replaced the β5-β6 loop of OXA-48 with the corresponding loop from OXA-18.
  • Enzyme kinetics: determined kinetic parameters (kcat, Km) for substrate hydrolysis.
  • MIC determination: assessed the impact of the engineered enzyme on antibiotic resistance.
  • Structural analysis: employed X-ray crystallography and molecular modeling to understand loop conformation and substrate binding.

Main Results:

  • The hybrid enzyme OXA-48Loop18 retained the ability to hydrolyze both carbapenems and ESCs.
  • Kinetic analysis showed a lower catalytic efficiency (kcat) for carbapenem hydrolysis compared to native OXA-48, but expanded ESC hydrolysis.
  • Structural studies indicated that the grafted loop adopts a conformation allowing binding of bulkier substrates, expanding the enzyme's hydrolytic profile.
  • The observed changes are attributed to both amino acid sequence and backbone conformation of the β5-β6 loop.

Conclusions:

  • The β5-β6 loop plays a crucial role in determining the substrate selectivity of OXA-48-like β-lactamases.
  • Localized modifications, including loop grafting, can significantly alter or expand the functional capabilities of β-lactamases, highlighting their inherent plasticity.
  • Understanding these structure-function relationships provides insights into the evolution of antibiotic resistance mechanisms.