Purification and characterization of a thermophilic NAD+-dependent lactate dehydrogenase from Moorella thermoacetica

Abstract

Oxidation of lactate under anaerobic dark fermentative conditions poses an energetic problem. The redox potential of the lactate/pyruvate couple is too electropositive to reduce the physiological electron carriers NAD(P)⁺ or ferredoxin. However, the thermophilic, anaerobic, and acetogenic model organism Moorella thermoacetica can grow on lactate but was suggested to have a NAD⁺-dependent lactate dehydrogenase (LDH), based on enzyme assays in cell-free extract. LDHs of thermophilic and anaerobic bacteria are barely characterized but have a huge biotechnological potential. Here, we have purified the LDH from M. thermoacetica by classical chromatography. Lactate-dependent NAD⁺ reduction was observed with high rates. Electron bifurcation was not observed. At pH 8 and 65 °C, the LDH had a specific activity of 60 U·mg^-1 for lactate oxidation, but NADH-driven pyruvate reduction was around four times faster with an activity of 237 U·mg^-1. Since lactate formation is preferred by the enzyme, further modifications of the LDH can be suggested to improve the kinetics of this enzyme making it a promising candidate for biotechnological applications.

Keywords: acetogen; anaerobe; lactate; purification; thermophile.

Fig. 1

Genetic organization and comparison of the LDH from Moorella thermoacetica to LDHs from other bacteria. The potential LDH gene is colored in blue. The LDH gene is surrounded by the genes Mothe_c18530 and Mothe_c18550, encoding for a putative transposase and a transcriptional regulator (A). The LDH was compared to the LDH from Lactobacillus acidophilus, Streptococcus equinus, Bifidobacterium bifidum, Bacillus subtilis, Thermotoga maritima, Bacillus caldolyticus, and Geobacillus stearothermophilus. The identity is given in percent. □, Rossmann fold NAD(P)⁺ binding domain; Δ, fructose‐1,6‐bisphosphate‐binding domain; ■, active site (B). A structural overlay of the LDH from M. thermoacetica (purple), T. maritima (blue), and B. subtilis (yellow) indicates structural similarity (C).

Fig. 2

Purification of the LDH from Moorella thermoacetica. Proteins of the cytoplasm were separated by anion exchange chromatography using a NaCl gradient from 0 to 1 m NaCl over 200 mL. Fractions containing LDH activity are indicated by blue shading (A). Pooled fraction after separation by anion exchange chromatography were precipitated and further separated by hydrophobic interaction chromatography. Fractions containing LDH activity are indicated by blue shading (B). Pooled fraction after hydrophobic interaction chromatography were concentrated by ultrafiltration and further separated by size exclusion chromatography. The LDH eluted in a single peak (C, blue shading). Size exclusion chromatography column was calibrated using aprotinin (6.5 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), and conalbumin (75 kDa) as standard proteins, indicated by gray symbols (D). The elution behavior of the LDH is indicated by the red symbol.

Fig. 3

SDS/PAGE monitoring the purification process of the LDH from Moorella thermoacetica. Samples of the different purification steps were separated by SDS/PAGE (12%), and proteins were stained with Coomassie Brilliant Blue G250. Ten microgram of protein was applied to each lane. CE, cell‐free extract; CP, cytoplasm; M, prestained page ruler; P, pooled fractions from Phenyl‐ Sepharose; Q, pooled fractions from Q‐ Sepharose; S, pooled fractions from size exclusion Superose 6.

Fig. 4

Biochemical characterization of the LDH. All assays were performed in 1.8‐mL anoxic cuvettes containing an overall liquid volume of 1 mL and 5 μg protein and a 100% N₂ atmosphere. (A) lactate‐dependent NAD⁺ reduction. The assay contained 2 mm NAD⁺ in buffer A. (B) Temperature optimum of the LDH, the assay contained 2 mm NAD⁺ in buffer A at 40–70 °C. (C) pH optimum of the lactate‐dependent NAD⁺ reduction, the assay contained 2 mm NAD⁺ in buffer B1 at 65 °C. (D) Pyruvate‐dependent NADH oxidation of the LDH, the assay contained 2 mm NADH in buffer B2 at 65 °C. (E) K _m determination for NAD⁺, and the assay contained 0–2 mm NAD⁺ in buffer A at 65 °C. (F) K _m determination for l‐lactate. The assay contained 2 mm NAD⁺ and 0–100 mm l‐lactate in buffer A at 65 °C. (G) K _m determination for NADH. The assay contained 0–0.5 mm NADH and 1 mm pyruvate in buffer A at 65 °C. (H) K _m determination for pyruvate. The assay contained 0.5 mm NADH and 0–5 mm pyruvate in buffer A at 65 °C. The reactions were started by adding 20 mm l‐lactate or 5 mm pyruvate, if not otherwise stated (n = 3; SD).

Fig. 5

Scheme of lactate metabolism in Moorella thermoacetica. It is assumed that the endergonic reduction of NAD⁺ with lactate as electron donor becomes feasible by coupling this reaction to two electron‐bifurcating enzymes [6]. First, the electron‐bifurcating EtfABCX complex oxidizes NADH released by the oxidation of lactate to pyruvate and reduces MQ and Fd. Next, the electron‐bifurcating MTHFR reduces the intermediates methylene‐THF to methyl‐THF of the Wood–Ljungdahl pathway with MQ and NADH as reductants. Methyl‐THF is than further metabolized in the Wood–Ljungdahl pathway to acetate (adapted from Ref. [6]). The reduced Fd is used by ferredoxin‐dependent NADH‐dehydrogenase NDH‐1 to establish the chemiosmotic gradient. This gradient fuels the ATP synthase to generate ATP.

Fig. 6

Comparison of LDHs and EtfABCX complexes in different Moorella strains. The LDH from Moorella thermoacetica is compared to enzymes from different Moorella strains (A). Similarity is given in %. □, Rossmann fold NAD(P)⁺ binding domain; Δ, fructose‐1,6‐bisphosphate binding domain; ■, active site. The EtfABCX complex from M. thermoacetica and different Moorella strains is compared (B). Same color indicates similar genes, similarity is given in %; ●, FAD binding domain; □, Rossmann fold NAD(P)⁺ binding domain; ▲, quinone binding domain; ♦, 4Fe‐4S binding domain.