Fig 1.
Growth of glucarpidase producing bacteria.
1A. Bacterial growth on folate as the only carbon source LB/agar plate. (a) Before the bacterial growth and (b) the bacterial growth after several days. 1B. A bright-blue fluorescence under U.V. light. (a) The Folate media under UV light while (b) is the growing bacteria on folate plate showing the bright-blue fluorescence of lumazine-6-carboxylic acid resulting from folate hydrolysis by the growing strain. 1C. 1% Agarose gel electrophoresis of the cell extract of one isolate showing bright blue fluorescence (lumazine-6-carboxylic acid) under U.V. light, resulting from folate degradation by the isolated strain.
Fig 2.
MS of the protonated DAMPA is shown at 313.1m/z.
a) DAMPA H+ peak is the product of folate hydrolysis by the isolated strain. b and c) P1, P2 are DMPA H+ produced by recombinant CPG2s, new and Ps CPG2 respectively and F1, F2 are intact folate isolated from the media of both recombinant enzymes.
Fig 3.
Glucarpidase activity in the total soluble protein of the two novel glucarpidase producing bacteria.
MTX substrate solution and total soluble protein of Pseudomonas lubricants strain SF168 in presence of Zn2+ (filled ball) and in presence of Zn2+ and EDTA (diamond). MTX substrate solution and total soluble protein from Xenophilus sp. SN213 in the presence of Zn2+ (star), and in the presence of Zn2+ and EDTA (filled triangle), and control of MTX and enzyme buffer (square) are shown. The data in this figure indicates that both strains (Pseudomonas lubricans strain SF168 and Xenophilus sp. SN213) show CPG2 activity through methotrexate hydrolysis.
Fig 4.
Amino acids sequence alignment of glucarpidase genes.
the uppermost sequence encodes the glucarpidase from Pseudomonas sp. Strain RS-16 (12), the lower sequences encode the new glucarpidases isolated from Xenophilus azovorans SN213 and Stenotrophomonas sp SA, respectively (this work). Amino acid differences between the sequences are highlighted in red. Amino acids in blue are involved in the active site of the enzyme where two zinc ions and a bridging water molecule binds (30).
Fig 5.
CPG2 Protein expression of the gene isolated from Xenophilus azovorans SN213.
a. Coomassie blue staining of 10% SDS-PAGE of E. coli BL21(DE3)RIL-CPG2 protein expression at 37°C of the gene isolated from Xenophilus azovorans SN213. M is the PageRuler Prestained Protein Ladder (10 to 180 kDa), lanes 1 and 2 are the induced soluble and insoluble fractions respectively, and lanes 3 and 4 are the uninduced soluble and insoluble fractions respectively. b. Protein expression at 20°C of the gene isolated from Xenophilus azovorans. SN213. M is the prestained protein ladder, lanes 1 and 2 are the induced soluble and insoluble fractions respectively, and lanes 3 and 4 are the uninduced soluble and insoluble fractions respectively.
Fig 6.
Recombinant CPG2 activity on folate agar plate.
The activity of the isolated recombinant CPG2 in comparison to Ps CPG2 of Pseudomonas sp strain RS-16 and in the presence of negative control (vector only) on LB/KAN/CAM/IPTG/Folate in the presence of 0.2 mM ZnSO4.
Fig 7.
Purification of recombinant new glucarpidase relative to Ps CPG2.
Coomassie blue staining of a 10% SDS-PAGE gel. M; Size markers in kiloDaltons; M1 is PageRuler Prestained Protein Ladder (10 to 180 kDa) and M2 is SeeBlue Plus prestained standard (↱3 to 198 kDa). a) Xen CPG2 purification; lane 1 is total soluble fraction of glucarpidase after centrifugation; 2, flow through; 3–4, wash 1 and wash 5; 5–9, eluted fractions from the Ni-NTA column with 400 mM imidazole. b) Ps CPG2 purification; lanes 1, 2, 3 are total, flow through, wash, lanes 4–6 are elution fractions.
Fig 8.
Carboxypeptidase G2 activity is Zn2+ dependent.
MTX substrate solution and pure CPG2 in the presence of Zn2+ (star), control reaction of MTX substrate solution and buffer (filled triangle) and MTX substrate solution and pure CPG2 in the presence of Zn2+ and 10 mM EDTA (square) are shown. New isolated CPG2 is Zn2+ dependent.
Fig 9.
CD spectra and high voltage of Xen CPG2 and Ps CPG2.
A) Combined CD spectra data of Xen CPG2 and Ps CPG2 in molar ellipticity relative the wavelength in far UV region, the spectra obtained by dragging their spectral data over each other, where smooth 0 is molar ellipticity of Xen CPG2 and smooth 1 is for CD spectra of Ps CPG2. All Spectral data are corrected for the baseline buffer, B) represents the combined High voltage (HV) for both enzymes where average 0 is Xen CPG2 and average 1 is Ps CPG2. Also the table shows the calculated protein secondary structure of Xen CPG2 and Ps CPG2 by CDNN deconvolution analysis using their CD spectral data.
Fig 10.
Homology modeling of Xen CPG2.
A. Electrostatic surface presentation of the Xen CPG2 tetramer. Positive and negative charges are shown in blue and red, respectively. B. Color rendering of a Xen CPG2 monomer. Helix in cyan, b-sheet in pink, loops in brown and amino acids which differ from the model carboxypeptidase G2 are shown as yellow sticks. Rendering was performed using PyMol. C. Stereoview of the alignment of CPG2 Pseudomonas sp. Strain RS-16 (blue cartoon, PDB ID 1CG2) with the model of Xen CPG2 (green cartoon, RMS = 0.084 (374 to 374 atoms)).
Fig 11.
Antibody detection of newly isolated CPG2 relative to Ps CPG2.
A. Dot blot using anti His tag antibody and using anti Xen CPG2 antibody where 1, 2, and 3 are pure protein (Xen CPG2 and Ps CPG2) at concentrations (0.05, 0.1, and 0.2 mg/mL). B. Dot blot at different concentration of anti Xen CPG2 antibody where 1, 2, and 3 are blotting at dilutions 1:20 000, 1:10 000 and 1:3000 in blocking buffer. C. SDS-PAGE and Western blot analysis of the pure protein (Xen CPG2 and Ps CPG2) where M is PageRuler™ Unstained Protein Ladder (10–200 kDa), lanes 1, 2, 3 are 0.25, 0.1, and 0.05 mg/mL of Xen CPG2 and lanes 4, 5, 6 are the same series of protein concentrations of Ps CPG2.