Photoaffinity probe-enabled discovery of sennoside A reductase in Bifidobacterium pseudocatenulatum

Abstract

Sennoside A (SA), a typical prodrug, exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases. Hydrolases have been identified, but reductases remain unknown. By linking a photoreactive group to the SA scaffold, we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling (ABPP). From lysates of an active strain, Bifidobacterium pseudocatenulatum (B. pseudocatenulatum), 397 proteins were enriched and subsequently identified using mass spectrometry (MS). Among these proteins, chromate reductase/nicotinamide adenine dinucleotide (NADH) phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase/oxygen-insensitive NADPH nitroreductase (nfrA) was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource (UniProt) database screening. We also determined that recombinant nfrA could reduce SA. Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.

Keywords: Chemical biology; Gut bacteria; Photoaffinity labeling; Reductase; Sennoside A; Small molecule; Transformation.

Graphical abstract

Scheme 1

Activity-based protein profiling (ABPP) was used to probe sennoside A (SA) reductases. (A) Structure of the photoaffinity probe based on SA (SAP). (B) Workflow of probing SA reductases using ABPP. UV: ultraviolet; LC-MS: liquid chromatography-mass spectrometry.

Fig. 1

Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) could reduce sennoside A (SA). (A) Metabolic pathways of SA in bacterial strains and the derivatization of reductive products. (B, C) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of azomethyl derivatives of 8-glucosylrheinanthrone (B) and rheinanthrone (C).

Fig. 2

Activity of human nicotinamide adenine dinucleotide (phosphate) (NAD(P)H): quinone dehydrogenase 1 (hNQO1) to reduce sennoside A (SA) was verified. (A, B) The three-dimensional (3D) (A) and 2D (B) binding mode of hNQO1 and SA. (C–E) The production of 8-glucosylrheinanthrone (C), rhein-8-O-β-d-glucoside (D), and total metabolites of SA (E) changed over time after incubating hNQO1 with SA. (F, G) The amounts of residual SA (F) and the reductive products (G) after incubating hNQO1 with SA and dicoumarol (DIC). ^∗∗∗P < 0.001, compared with the control group. FAD: flavin adenine dinucleotide.

Fig. 3

The photoaffinity probe based on sennoside A (SA) (SAP) could be reduced by human nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) quinone dehydrogenase 1 (hNQO1) and label proteins under ultraviolet (UV) irradiation. (A) Two inferred reductive products of SAP and derivatives of the products. (B, C) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the reductive products of SAP catalyzed by hNQO1 at 0.5 (B) and 12 h (C) (experimental group I and control group II). (D) Workflow of in-gel fluorescence analysis. (E) Evaluation of labeling effects of SAP by in-gel fluorescence. OVA: ovalbumin; CBB: Coomassie brilliant blue; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Fig. 4

The potent sennoside A (SA) reductases in Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) were labeled by the photoaffinity probe based on SA (SAP). (A) Labeling of live bacterial cells or bacterial lysates with SAP. (B) Labeling of bacterial lysates with SAP under a series of incubation time. (C) Labeling of bacterial lysates with a series of dose of SAP. (D, E) Two digestion procedures for proteins enriched (D) and Venn diagram analysis of the corresponding results (E). CBB: Coomassie brilliant blue; MW: molecular weight; SDS: sodium dodecyl sulfate; LC-MS: liquid chromatography-mass spectrometry.

Fig. 5

Chromate reductase/nicotinamide adenine dinucleotide (NADH) phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase/oxygen-insensitive NADPH nitroreductase (nfrA) was selected from the identified proteins and validated as a sennoside A (SA) reductase. (A) Bioinformatic analysis of 397 proteins labeled by the photoaffinity probe based on SA (SAP). (B, C) The three-dimensional (3D) (B) and 2D binding mode (C) of nfrA and SA. (D, E) The amount of azomethine derivatives of 8-glucosylrheinanthrone (D) and rhein-8-O-β-d-glucoside (E) changed over time in the control and nfrA experimental groups. ^∗∗P < 0.01 and ^∗∗∗∗P < 0.0001, compared with the control group. (F, G) Kinetic interaction between SA and nfrA (F) was performed using surface plasmon resonance (SPR) analysis. The fitted curves were mapped for different concentrations of SA (G) binding to nfrA using kinetic analysis. GO BP: Gene Ontology (GO) biological process; GO MF: GO molecular function; rRNA: ribosomal RNA; GO CC: GO cellular component; KEGG: Kyoto Encyclopedia of Genes and Genomes; NADP: nicotinamide adenine dinucleotide phosphate.