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

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 CO₂ 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.