Thermostable and O2-Insensitive Pyruvate Decarboxylases from Thermoacidophilic Archaea Catalyzing the Production of Acetaldehyde

Abstract

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO₂. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, T_opt = 80 °C) and Sulfolobus acidocaldarius (Sa, T_opt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.

Keywords: Saccharolobus solfataricus; Sulfolobus acidocaldarius; archaea; ethanol fermentation; hyperthermophiles; pyruvate decarboxylase; pyruvate:ferredoxin oxidoreductase.

Figure 1

Pathways for ethanol production from pyruvate. Left side, two-step pathways catalyzed by PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase); Right side, three-steps pathway catalyzed by POR (pyruvate:ferredoxin oxidoreductase), AcDH (CoA-dependent acetaldehyde dehydrogenase), and ADH (alcohol dehydrogenase).

Figure 2

Analyses of purified bifunctional POR/PDCs from S. solfataricus (A) and S. acidocaldarius (B) using SDS-PAGE (12.5%). (A) Lane 1, 10 μg of CFE; lane 2, 20 µg of DEAE fraction; lane 3, 18 µg of HAP fraction; lane 4, 1.5 µg of purified enzyme; lane 5, BLUeye pre-stained protein ladder. (B) Lane 1, 12 μg of CFE; lane 2, 33 µg of DEAE fraction; lane 3, 22 µg of HAP fraction; lane 4, 0.8 µg of purified enzyme; lane 5, BLUeye pre-stained protein ladder.

Figure 3

Temperature dependence of the POR and PDC activities of the bifunctional POR/PDCs from S. solfataricus (A) and S. acidocaldarius (B). POR and PDC enzyme activities were determined over a temperature range from 40 to 90 °C. The assay mixture of POR contains 100 mM sodium phosphate pH 8.0, 5 mM pyruvate, 100 µM CoA, 1 mM benzyl viologen and approximately 50 µM sodium dithionite. The PDC assay mixture was 100 mM sodium phosphate at pH 8.0, 10 mM pyruvate, and 100 µM CoA. The relative activities of 100% are equal to the highest specific activities (12.1 U/mg for SsPOR, 0.069 U/mg for SsPDC, 8 U/mg for SaPOR, and 0.057 U/mg for SaPDC activities). The insets show the Arrhenius plots.

Figure 3

Temperature dependence of the POR and PDC activities of the bifunctional POR/PDCs from S. solfataricus (A) and S. acidocaldarius (B). POR and PDC enzyme activities were determined over a temperature range from 40 to 90 °C. The assay mixture of POR contains 100 mM sodium phosphate pH 8.0, 5 mM pyruvate, 100 µM CoA, 1 mM benzyl viologen and approximately 50 µM sodium dithionite. The PDC assay mixture was 100 mM sodium phosphate at pH 8.0, 10 mM pyruvate, and 100 µM CoA. The relative activities of 100% are equal to the highest specific activities (12.1 U/mg for SsPOR, 0.069 U/mg for SsPDC, 8 U/mg for SaPOR, and 0.057 U/mg for SaPDC activities). The insets show the Arrhenius plots.

Figure 4

pH dependence of the POR and PDC activities of the bifunctional POR/PDCs from S. solfataricus (A) and S. acidocaldarius (B). POR activities were assayed using 5 mM pyruvate, 1 mM methyl viologen, 100 µM CoA, and approximately 50 µM sodium dithionite at 80 °C. The following buffers (100 mM) were used: sodium phosphate (pH 4.9, 5.9, and 6.9), glycine (pH 7.6, 8.1 and 8.6), and CAPS (pH 9.5, 10.5, and 11.5). PDC activities were measured using 10 mM pyruvate and 100 µM CoA at 80 °C. The following buffers were used: sodium phosphate (pH 6.9, and 7.9), glycine (pH 7.6, and 8.6), and CAPS (pH 10.5, and 11.5). The relative activities of 100% are equal to the highest specific activities (2.2 U/mg for SsPOR, 0.16 U/mg for SsPDC, 0.55 U/mg for SaPOR and 0.052 U/mg for SaPDC activity).

Figure 4

pH dependence of the POR and PDC activities of the bifunctional POR/PDCs from S. solfataricus (A) and S. acidocaldarius (B). POR activities were assayed using 5 mM pyruvate, 1 mM methyl viologen, 100 µM CoA, and approximately 50 µM sodium dithionite at 80 °C. The following buffers (100 mM) were used: sodium phosphate (pH 4.9, 5.9, and 6.9), glycine (pH 7.6, 8.1 and 8.6), and CAPS (pH 9.5, 10.5, and 11.5). PDC activities were measured using 10 mM pyruvate and 100 µM CoA at 80 °C. The following buffers were used: sodium phosphate (pH 6.9, and 7.9), glycine (pH 7.6, and 8.6), and CAPS (pH 10.5, and 11.5). The relative activities of 100% are equal to the highest specific activities (2.2 U/mg for SsPOR, 0.16 U/mg for SsPDC, 0.55 U/mg for SaPOR and 0.052 U/mg for SaPDC activity).

Figure 5

The pyruvate and CoA dependency of the PDC activities of the bifunctional POR/PDCs from S. solfataricus (A,B) and S. acidocaldarius (C,D). The CoA dependency ((A,C) 0.0 to 100 µM in the presence of 100 mM sodium phosphate [pH 7.9] and 10 mM pyruvate) and pyruvate dependency ((B,D) 0.0 to 10 mM in the presence of 100 mM sodium phosphate [pH 7.9] and 100 µM CoA) were performed at 80 °C.

Figure 5

The pyruvate and CoA dependency of the PDC activities of the bifunctional POR/PDCs from S. solfataricus (A,B) and S. acidocaldarius (C,D). The CoA dependency ((A,C) 0.0 to 100 µM in the presence of 100 mM sodium phosphate [pH 7.9] and 10 mM pyruvate) and pyruvate dependency ((B,D) 0.0 to 10 mM in the presence of 100 mM sodium phosphate [pH 7.9] and 100 µM CoA) were performed at 80 °C.