Article Figures & Data
Figures
- FIG 1
Route for the directed evolution of UPO1 toward functional expression and improved activity. New mutations are depicted as stars and accumulated mutations as squares. Mutations in the mature PaDa-I mutant and their origins are boxed. The signal peptide is represented in dark red and the mature protein in red. In the parental n-UPO1, the glycosylation sites (Asn11, Asn141, Asn161, Asn182, Asn286, and Asn295) are represented as blue stars, the Phe triad (Phe69, Phe121, and Phe199) involved in the binding of aromatic substrates is marked with green arrows, and the acid-base pair for peroxide cleavage (Glu196 and Arg189) is indicated with blue arrows. TAI represents the improvement in UPO1 activity detected in S. cerevisiae microcultures for each mutant compared with the parental n-UPO1. Thermostability (T50) was estimated from culture supernatants (Fig. 2B). The breakdown of the TAI into specific activity and expression is shown in Fig. 3. The dashed arrows indicate the parental types used for each round of evolution. In the 3rd generation, “a” indicates the offspring obtained by IvAM of 12C12 and “b” indicates the offspring obtained by MORPHING in the signal peptide of 12C12. In the 4th generation, “c” indicates the triple mutant at the signal peptide constructed using I13D3 as a template and “d” the offspring obtained by mutagenic PCR and shuffling of parents I13D3, M5D2, and M4D8. n.d., not determined; n.m., not measurable. (See also Table S1 in the supplemental material.)
- FIG 2
Biochemical characterization. (A) Spectroscopic characteristics of wtUPO1 (dashed line) and the PaDa-I mutant (solid line). AU, arbitrary units. (B) Thermostability (T50) of PaDa-I and different parental types. Each point represents the mean and standard deviation of 3 independent experiments. 1G to 5G, 1st generation to 5th generation. (C and D) pH activity profiles for wtUPO1 (triangles) and PaDa-I (squares). Activities were measured in 100 mM citrate/phosphate/borate buffer at different pH values with 2 mM H2O2 and 0.3 mM ABTS (C) or with 1 mM H2O2 and 1 mM NBD (D). UPO1 activity was normalized to the optimum activity value, and each point represents the mean and standard deviation of 3 independent experiments.
- FIG 3
Breakdown of specific activity and functional expression. A fusion gene containing the evolved signal peptide (n*) attached to the native mature UPO1 was engineered. The n-UPO1, n*-UPO1, and PaDa-I variants were produced on a large scale, and their TAIs were measured. n*-UPO1 and PaDa-I were purified, and their specific activities were calculated. n* enhanced functional expression ∼27-fold, whereas mutations in mature PaDa-I resulted in an ∼120-fold increase in total activity. The 3,250-fold increase in the total activity of PaDa-I was broken down as a 3.6-fold improvement in specific activity and a 1,114-fold improvement in functional expression.
- FIG 4
Activity and stability in organic cosolvents. (A and B) The relative activities of wtUPO1 (A) and the PaDa-I mutant (B) in organic cosolvents were assessed with 2 mM H2O2 and 0.3 mM ABTS in 100 mM sodium phosphate/citrate buffer (pH 4.4) containing the corresponding concentration of cosolvent. (C and D) The stabilities of wtUPO1 (C) and the PaDa-I mutant (D) after incubation for 48 h in 50% organic cosolvents were assessed by incubating enzyme samples in 100 mM potassium phosphate buffer (pH 7.0) containing 50% (vol/vol) organic cosolvent in screw-cap vials. After 48 h, aliquots were removed and analyzed in an activity assay with 2 mM H2O2 and 0.3 mM ABTS in 100 mM sodium phosphate/citrate buffer (pH 4.4). (E and F) The stabilities of wtUPO1 (E) and the PaDa-I mutant (F) at high concentrations of organic cosolvents were assessed after 5 h of incubation in increasing concentrations of cosolvents and incubating enzyme samples at 20°C in 100 mM potassium phosphate buffer (pH 7.0) containing increasing concentrations (vol/vol) of organic cosolvent (60 to 90%). After 5 h, aliquots were removed and analyzed in the activity assay, as described for panels C and D. Residual activities were expressed as percentages of the original activity at the corresponding concentration of organic cosolvent. The error bars indicate standard deviations.
- FIG 5
Mutations in evolved UPO1. A molecular model using as the template the A. aegerita crystal structure (PDB code 2YOR) was prepared to map the mutations. Shown are details of the 5 mutations (green) in the PaDa-I mutant (B, D, and F) compared with the corresponding residues (yellow) in the native UPO1 (A, C, and E). The dashed lines indicate distances (in Å) from the surrounding residues. Phe residues delimiting the active site are highlighted in pink and the Cys36 axial ligand in light blue. The Fe3+ of heme is shown as a red sphere and the structural Mg2+ as a pink sphere. (See also Table S2 and Fig. S2 in the supplemental material.)
Tables
- TABLE 1
Biochemical features of the wild type and the evolved UPO variant
Biochemical or spectroscopy featureh Value wtUPO1e PaDa-I mutantf Mass (Da)a 46,000 52,000 Mass (Da)b NDg 51,100 Mass (Da)c 35,942 35,914 Glycosylation degree (%) 22 30 Thermal stability (T50) (°C)d 53 55 pI 4.9–5.7 5.5 Optimum pH for ABTS 4.0 4.0 Optimum pH for DMP 7.0 6.0 Optimum pH for NBD 6.5 6.0 Rz (A418/A280) 2.4 1.8 Soret region (nm) 420 418 CT1 (nm) 572 570 CT2 (nm) 540 537 ↵a Estimated by SDS-PAGE.
↵b Estimated by MALDI-TOF mass spectrometry.
↵c Estimated from the amino acid composition.
↵d Estimated from purified variants.
↵e wtUPO1, UPO1 wild type expressed in A. aegerita.
↵f PaDa-I mutant, the ultimate variant of the whole evolution process in S. cerevisiae (containing the evolved signal peptide [n*] plus the evolved UPO1) (see Fig. S1 in the supplemental material).
↵g ND, not determined.
↵h CT1 and CT2, charge transference bands 1 and 2, respectively.
- TABLE 2
Kinetic parameters of wild-type, recombinant, and evolved UPO variants
Substrate Kinetic constant Valuea wtUPO1 n*-UPO1 PaDa-I ABTS Km (mM) 0.025 ± 0.002 0.027 ± 0.005 0.048 ± 0.004 kcat (s−1) 221 ± 6 45.0 ± 2.7 395 ± 13 kcat/Km (mM−1 s−1) 8,800 ± 692 1,600 ± 37 8,200 ± 598 NBD Km (mM) 0.684 ± 0.207 0.782 ± 0.352 0.483 ± 0.095 kcat (s−1) 219 ± 25 31.7 ± 6.1 338 ± 22 kcat/Km (mM−1 s−1) 320 ± 64 38.0 ± 11 700 ± 99 Benzyl alcohol Km (mM) 1.90 ± 0.11 1.10 ± 0.23 2.47 ± 0.32 kcat (s−1) 329 ± 7 44.8 ± 3.1 307 ± 15 kcat/Km (mM−1 s−1) 174 ± 7 41.0 ± 6.3 124 ± 11 Veratryl alcohol Km (mM) 5.20 ± 0.31 5.30 ± 0.82 7.9 ± 0.7 kcat (s−1) 88 ± 2 15.2 ± 1.1 121 ± 5 kcat/Km (mM−1 s−1) 17 ± 0.7 2.9 ± 0.25 15 ± 0.9 H2O2 Km (mM) 1.37 ± 0.16 0.69 ± 0.20 0.49 ± 0.06 kcat (s−1) 290 ± 15 40.9 ± 3.8 238 ± 8 kcat/Km (mM−1 s−1) 211 ± 15 59.0 ± 12.3 500 ± 42 ↵a ABTS kinetic constants for UPO1 were estimated in 100 mM sodium citrate/phosphate buffer, pH 4.4, containing 2 mM H2O2 and those for the rest of the substrates in 100 mM potassium phosphate buffer, pH 7.0, containing 2 mM H2O2 (benzyl and veratryl alcohols) or 1 mM H2O2 (NBD). H2O2 kinetic constants were estimated using benzyl alcohol as a reducing substrate under the corresponding saturated conditions. wtUPO1, UPO1 wild-type expressed in A. aegerita; n*-UPO, native UPO1 fused to the evolved signal peptide for secretion in S. cerevisiae; PaDa-I mutant, the ultimate variant of the whole evolution process in S. cerevisiae (containing the evolved signal peptide plus the evolved UPO1).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Strategies and mutations from the directed UPO1 evolution (Table S1), mutations in the mature PaDa-I variant (Table S2), primer list (Table S3), purification and glycosylation degrees (Fig. S1), overview of all the residues mutated by evolution (Fig. S2), and supplemental materials and methods (construction of fusion genes, high-throughput dual-screening assay development, large-scale enzyme production and purification protocols, MALDI-TOF-MS analysis, pI determination, protocols for steady-state kinetic constants, pH activity profiles, and protein modeling).
PDF, 620K
- Supplemental file 1 -
