Work

Production, crystallographic studies, and functional profiling of γ-carbonic anhydrase from the probiotic Limosilactobacillus reuteri: In vitro and cell-based insights

Year: 2025

Type: article

Abstract: Limosilactobacillus reuteri (formerly Lactobacillus reuteri ) is a probiotic bacterium involved in maintaining gut microbiota balance and modulating immune response. In this study, for the first time, we report the recombinant production and kinetic characterization of its γ-class carbonic anhydrase (CA, EC 4.2.1.1), referred to as LreCAγ. The enzyme catalyzes CO 2 hydration with an efficiency comparable to that of other bacterial γ-CAs, although lower than that of α-hCA II. X-ray crystallization studies shed light on the enzyme structure, and inhibition studies with anions, sulfonamides, and related compounds revealed that LreCAγ is less susceptible to inhibition compared to the γ-class CA from Vibrio cholerae , used for comparison. Otherwise, activation assays with selected amines and amino acids identified the two enantiomers of His ( 25 and 26 ) as the most potent LreCAγ activators. Stereochemistry had a minimal impact on activity, except for L -Phe ( 27 ), which was twice as potent as its D -enantiomer ( 28 ). To assess the biological effects of CA modulation, E. coli DH5α, which expresses several CAs, was used as a model organism. CA activators were tested alone and in combination with the pan-CA inhibitor acetazolamide ( AAZ ), revealing CA-dependent effects on bacterial growth. Additionally, selected CA activators were evaluated for their effects on human macrophages and intestinal epithelial cells, with L -Trp ( 31 ) attenuating LPS-induced activation and exhibiting good biocompatibility in normal intestinal cells. Taken together, these results underscore the feasibility of targeting LreCAγ activation as a strategy to enhance probiotic efficacy. • We produced and characterized the γ-class CA of Limosilactobacillus reuteri • Activation assays identified His as the most potent LreCAγ activator • Stereochemistry had a minimal impact on CA activation activity • CA activators were evaluated on human macrophages and intestinal epithelial cells • The first crystal structure of LreCAγ was obtained

Source: European Journal of Medicinal Chemistry

DOI: 10.1016/j.ejmech.2025.118291