Workflow the algorithm utilizes to generate predictions for a given enzyme or an organism

Algorithm testing using positive and negative control examples. Positive controls were chosen among known flavonoid-metabolizing enzymes and negative control enzymes were chosen from glycolysis pathways as they are not expected to metabolize flavonoids.

Predicted reaction types for the tested organisms mouse ear cress, F. plautii, E. coli, human and mouse. Correlation values (R-value) between the sequence similarity and RClass similarity of predicted enzymes, and statistical significance of the correlation value (p-value) have been reported in the last two columns.

Comparison of sequence similarity and R-class similarity in predicted enzymes for A) di/hydroxylation in mouse ear cress, B) de/hydrogenation in human, C) O-deglycosylation of in F. plautii and D) di/hydroxylation in E. coli

The p-values and R-values are calculated in Python using the pearsonr function from the SciPy package.

A) Proposed tilianin microbial metabolism pathway. B) Concentrations of parent compounds and their predicted immediate metabolites in the bacterial monocultures of B. animalis, B. coccoides, F. plautii, E. coli and microbe-free medium.

Results have been represented in bar graphs and displayed together in a matrix format, where the columns represent the microbes, and the rows represent the parent compound that is spiked into the culture.

Concentrations of tilianin, acacetin and apigenin in the tilianin-treated co-culture of A) B. animalis and B. coccoides, and B) B. animalis & F. plautii after 24 and 48 hours of incubation.

Tilianin metabolism pathway predicted by BioNavi-NP using the algorithm’s default modes. The outputs were redrawn to provide higher resolution for the predicted compounds. The most plausible pathway is colored in red, which overlaps with the pathway proposed in this work. The calculated cost of each reaction step is reflected by the confidence score where lower score means higher reaction probability.

PC-12 response too 200 μM H2O2 stress treatment for 4 hours following 1 hour pretreatment with tilianin, pathway metabolites (acacetin, apigenin, naringenin), or control conditions.

A. Viability was detected using calcein violet and shown as fraction of the untreated condition. B. The ROS level was assessed using DCFDA shown as a fraction of the untreated condition. N = 3 (figure shown is representative of all experiments); n = 4; *P < 0.05.

Example input and output from the tool developed in this work

We used chalcone synthase enzyme with the EC number as an input. The output suggests that this input enzyme can perform ring cleavage reaction on flavonoids and their metabolites, which aligns with our findings from Chapter 2.

Concentrations of possible microbial metabolites of apigenin and naringenin detected in the apigenin treated B. animalis monoculture.

Area under the curve for the detected apigenin and naringenin metabolites. Double sterisk (**) indicates significant difference (p < 0.005) compared to the vehicle control (no apigenin).

Tabulated flavonoids and their derivatives (KEGG COMPOUND numbers)

Tabulated enzymes from the chosen flavonoids and their derivatives

Enzymes that catalyze reactions which require molecular oxygen

Master RClass similarity table with a cutoff value of 0.8