A Versatile Aldehyde: Ferredoxin Oxidoreductase from the Organic Acid Reducing Thermoanaerobacter sp. Strain X514

Abstract

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AOR_X514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AOR_X514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AOR_X514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Keywords: AOR-ADH pathway; aldehyde:ferredoxin oxidoreductase; ethanol; organic acid reduction; thermoanaerobacter.

Figure 1

Putative function of aldehyde:ferredoxin oxidoreductase (AOR) in aldehyde detoxification. Reductive detoxification of accumulated aldehydes as product of amino acid ((a); after [17,18]) or alcohol catabolism ((b); after [14,19]). AT: aminotransferase; DC: decarboxylase; ADH: alcohol dehydrogenase.

Figure 2

Pathways for enzymatic carboxylic acid reduction. (a) Aldehyde:ferredoxin oxidoreductase and alcohol dehydrogenase involving (AOR-ADH) pathway for direct reduction of carboxylic acids to alcohols. Note that the pathway also reduces acetate produced from pyruvate oxidation in sugar fermentation. (b) Activation of carboxylic acids through acetate kinase (ACK) and phosphotransacetylase (PTA) to their acyl-CoA thioesters, and subsequent reduction by aldehyde dehydrogenase (ALDH) and ADH.

Figure 3

Separation of purified AOR_X514 with C-terminal His-Tag by polyacrylamide gel electrophoresis (PAGE). C: 5 µg cell free MB014 extract before affinity chromatography; F: 5 µg flow-through of all proteins not binding to the His-tag column; E: 2 µg eluate containing AOR-His, eluted with 150 mM imidazole were analyzed (a) on a 10% SDS-polyacrylamide gel stained with Coomassie blue, (b) used for Western blot with specific anti-AOR antibodies and (c) on a 10% native polyacrylamide gel stained with Coomassie blue (c, left side) and used for Western blot with specific anti-AOR antibodies (c, right side). For SDS PAGE, NEB Color Prestained Protein Standard Broad Range (New England BioLabs) marker and for native gel Serva Native Marker (Serva) was loaded onto the gel.

Figure 4

Temperature-dependent aldehyde oxidation activity of AOR_X514. (a) AOR activity was measured between 10 °C and 90 °C (dark grey circles) and between 65 °C and 95 °C (grey triangles) as acetaldehyde-dependent reduction of BV (ε₆₀₀ = 7.4 mM⁻¹ cm⁻¹). The reaction mixture contained in 50 mM TRIS, pH 7.5 with 1.2 mM BV, 12 µg (dark grey circles) or 15 µg (light grey squares) AOR_X514 and 1.2 mM acetaldehyde. AOR-His from two independent purifications was used, the average represents four technical replicates. The specific activities were normalized to the mean value of the activity at 65 °C. (b) Thermostability of AOR at 65 °C (dark grey circles), 75 °C (grey triangles) and 85 °C (light grey squares). AOR activity was measured in 50 mM TRIS with 2.4 mM benzyl viologen, 14 µg AOR-His and 1.2 mM acetaldehyde at 65 °C. The average represents three to five experiments, activities were normalized to the enzyme activity before heat treatment.

Figure 5

pH- and oxygen-dependent aldehyde oxidation activity of AOR_X514. (a) AOR activity was measured between pH 5.5 and pH 11.5 with 0.2 mM benzyl viologen (BV, circles) and between pH 5.5 and pH 9.0 (triangles) with 2 mM BV at 65 °C, with 14 µg AOR-His and 1.2 mM acetaldehyde. The buffers used were at concentrations of 50 mM, MES (5.5, 6.0, 6.5), MOPS (6.5, 7.0, 7.5), TRIS (7.5, 8.0, 8.5, 9.0), CHES (8.5, 9.0, 9.5, 10.0) and CAPS (10.0, 10.5, 11.0, 11.5) were used (from lightest to darkest). The average represents four technical replicates. The specific activities were normalized to the mean value of 50 mM TRIS, pH7.5. (b) AOR_X514 was exposed to air and the activity was measured regularly. Specific AOR activity was measured in 50 mM TRIS at pH 7.5 with 2.4 mM BV, 17 µg AOR_X514 and 1.2 mM acetaldehyde at 65 °C. The average (filled circle) represents five separate experiments (empty circles), activities were normalized to the enzyme activity before air exposure.

Figure 6

Substrate specificity of AOR-His. AOR activity was measured as aldehyde oxidation at 65 °C with 2 mM BV, 8 to 14 µg AOR-His and 2 mM, 1 mM, 0.1 mM or 0.01 mM aldehyde, as depicted. For each aldehyde the activity with 1 mM, 0.1 mM and 0.01 mM were tested, when the highest activity was with 1 mM, 2 mM aldehyde was tested additionally. The highest activity per aldehyde is represented in the figure. The average represents three separate experiments; activities were normalized to the enzyme activity with acetaldehyde.

Figure 7

Ferredoxin-dependent AOR activity. AOR activity was measured at 65 °C as acetaldehyde-dependent Fd reduction at 430 nm in 50 mM TRIS buffer at pH 7.5. (a) 20 µM His- TKV_c16450 was used as electron acceptor (b) 20 µM His-TKV_c09620 was used as electron acceptor, 50 µg AOR_X514 (E) were added and the reaction was started with the addition of 1 mM acetaldehyde (aa). (c) Between 4 to 212 µM His-TKV_c09620 were used as electron acceptor and 8 to 20 µg AOR-His, and 1 mM acetaldehyde. Three independent experiments with one to three technical replicates (grey circles, light grey triangles and dark grey diamonds).