Carboxypeptidase G and pterin deaminase metabolic pathways degrade folic acid in Variovorax sp. F1

Abstract

Background: Folic acid (FA) is a synthetic vitamin (B₉) and the oxidized form of a metabolic cofactor that is essential for life. Although the biosynthetic mechanisms of FA are established, its environmental degradation mechanism has not been fully elucidated. The present study aimed to identify bacteria in soil that degrade FA and the mechanisms involved.

Results: We isolated the soil bacterium Variovorax sp. F1 from sampled weed rhizospheres in a grassland and investigated its FA degradation mechanism. Cultured Variovorax sp. F1 rapidly degraded FA to pteroic acid (PA), indicating that FA hydrolysis to PA and glutamate. We cloned the carboxypeptidase G (CPG) gene and found widely distributed paralogs within the Variovorax genus. Recombinant CPG preferred FA and deaminofolic acid as substrates, indicating its involvement in FA degradation by Variovorax. Prolonged culture of Variovorax sp. F1 resulted in decreased rates of deaminofolic acid (DFA) and deaminopteroic acid (DPA) accumulation. This indicated that the deamination reaction also comprised a route of FA degradation. We also identified an F1 gene that was orthologous to the pterin deaminase gene (Arad3529) of Agrobacterium radiobacter. The encoded protein deaminated FA and PA to DFA and DPA, which was consistent with the deamination activity of FA and PA in bacterial cell-free extracts.

Conclusion: We discovered that the two enzymes required for FA degradation pathways in isolates of Variovorax sp. F1 comprise CPG and pterin deaminase, and that DFA and PA are intermediates in the generation of DPA.

Keywords: Deaminofolic acid; Pterin deaminase; Pteroic acid; Variovorax; Vitamin B9.

Fig. 1

Isolation of FA degrading bacteria. A Growth of eight isolates on M9-FA agar plates at 28 °C for 48 h. B Degradation of FA to PA by isolates cultured in M9-FA medium at 28 °C for 48 h. Filled bars, FA; unfilled bars, PA. Error bars, standard errors of means (n = 3, p < 0.05). *P < 0.05 (control FA vs. accumulated PA). C Nucleotide sequences of selected strains were aligned, and phylogenetic trees were constructed by neighbor-joining method [33] using MEGA X software [34]. Numbers along branches indicate 1000 bootstrap replicates D Time-dependent decomposition of FA by typical culture of Variovorax sp. F1 in M9-FA medium at 28 °C for 48 h. ●, FA; ■, PA; ▲, DFA; ◆, DPA. Bars, cell growth estimated as total cellular proteins. Error bars, standard errors of means (n = 3). DFA, deaminofolic acid; DPA, deaminopteroic acid; FA, folic acid; PA, pteroic acid

Fig. 2

Proposed FA degradation pathway in Variovorax sp. F1. Schematic model of FA degradation by Variovorax sp. F1. ABG, N-(4-aminobenzoyl)-L-glutamic acid; CPG, carboxypeptidase G; DFA, deaminofolic acid; DPA, deaminopteroic acid; FA, folic acid; PA, pteroic acid; PABA, p-aminobenzoic acid; PDA, pterin deaminase

Fig. 3

Phylogenetic relationship of CPG from Variovorax sp. F1. Amino acid sequences of predicted CPG from selected strains were aligned and phylogenetic trees were constructed by neighbor-joining [33] using MEGA X software [34]. Numbers along branches indicate 1000 bootstrap repeats. Gene IDs are shown after strain names in parenthesis

Fig. 4

Enzymatic properties of rCPG derived from Variovorax sp. F1. A SDS-PAGE gel. Lanes: M, molecular weight marker; rCPG, Recombinant CPG (1.8 μg) from Variovorax sp. F1 strain. B–D HPLC analyses of reactions containing 5 μg mL^− 1 rCPG and various substrates at 30 °C for 15 min. Substrates (5 mM) each are B FA; C DFA; D ABG. Traces: 1, substrates; 2, products; 3, reaction. E Specific activities of rCPG against FA, DFA, and ABG. Error bars indicate standard errors of means (n = 3). *P < 0.05. ABG, N-(4-aminobenzoyl)-L-glutamic acid; DFA, deaminofolic acid; DPA, deaminopteroic acid; FA, folic acid; PA, pteroic acid; rCPG, recombinant carboxypeptidase G

Fig. 5

Phylogenetic relationships of deaminase from Variovorax sp. F1 PDA. Amino acid sequences of putative hydrolases that catalyze non-peptide carbon-nitrogen bond cleavage (EC 3.5) from V. paradoxus S110 were aligned, and phylogenetic trees were constructed by neighbor-joining [33] using MEGA X software [34]. Numbers along branches indicate 500 bootstrap repeats. Gene IDs are color-coded according to their enzyme families as: hydrolases acting on linear amides (red), cyclic amides (blue), linear amidines (green), cyclic amidines (yellow), and nitriles (gray)

Fig. 6

Enzymatic properties of rPDA from Variovorax sp. F1. A SDS-PAGE gel. Lanes: M, molecular weight marker; rPDA, Recombinant PDA (2 μg) from Variovorax sp. F1 strain. B HPLC analyses of PDA reactions containing 0.01 μg mL^− 1 rPDA and 1 mM substrate (left, FA; right, PA) at 30 °C for 2 min. Traces: 1, substrates; 2, products; 3, reaction. C Initial velocity of rPDA reaction determined to calculate K_m and k_cat values of rPDA for FA (left) and PA (right) as substrates. Data were fitted to Michaelis-Menten equation

Fig. 7

Reactions of cell-free extract of Variovorax sp. F1 with various substrates. Folic acid (A) degradation of DFA (B), PA (C), and ABG (D) in cell-free extracts prepared from F1 strain cultured in M9-FA medium. Reactions proceeded in 50 mM Tris-HCl (pH 7.5) containing 0.2 mM ZnSO₄, cell-free extract (5 μg mL^− 1 protein) and substrates at 30 °C for 4 h. No DPA was degraded. ●, FA; ■, PA; ▲, DFA; ◆, DPA; ○, ABG; ◇, PABA. Error bars, standard errors of means (n = 3)