TY - JOUR
T1 - The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG
AU - Urbański, Linda J.
AU - Bua, Silvia
AU - Angeli, Andrea
AU - Emameh, Reza Zolfaghari
AU - Barker, Harlan R.
AU - Kuuslahti, Marianne
AU - Hytönen, Vesa P.
AU - Parkkila, Seppo
AU - Supuran, Claudiu T.
N1 - Funding Information:
This research was supported by funding from the Academy of Finland, the Jane & Aatos Erkko Foundation, and the Sigrid Juselius Foundation.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/6
Y1 - 2022/6
N2 - Abstract: We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, β, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only β- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s−1 and kcat/KM of 1.41 × 107 s−1 M−1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. Key points: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.
AB - Abstract: We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, β, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only β- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s−1 and kcat/KM of 1.41 × 107 s−1 M−1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. Key points: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.
KW - Alpha carbonic anhydrase
KW - In vitro inhibition
KW - Kinetics
KW - Lactobacillus rhamnosus
KW - Sulfonamide inhibition
U2 - 10.1007/s00253-022-11990-3
DO - 10.1007/s00253-022-11990-3
M3 - Article
C2 - 35612631
AN - SCOPUS:85130749701
SN - 0175-7598
VL - 106
SP - 4065
EP - 4074
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 11
ER -