The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui

Abstract

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd^2- ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg^-1 ) and ferredoxin (Fd; 27.2 U·mg^-1 ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein^-1 , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.

Keywords: extremophile; genetic engineering; homologous gene expression; protein production.

Fig. 1

Genetic organization, architecture, and cofactors of possible PFORs of Thermoanaerobacter kivui. The genome of T. kivui encodes for three different pfor clusters (A). Regardless of gene arrangements, PFORs maintain a basic composition of domains I, II, III, and VI, with domain V also present in most PFORs. Domain IV is present only in dimeric PFORs. Cartoons of the domain arrangement for the catalytic units of possible PFORs in T. kivui are shown (B). Domains are indicated with colored boxes, with their respective domain numbers inside. Black bars connecting domains indicate that the domains are found on the same polypeptide chain. The domains that bind TPP and [4Fe–4S] cluster are indicated at the top of each domain. VIT; vacuolar iron transporter.

Fig. 2

SDS/PAGE monitoring the purification process of PFOR1. Samples from the different purification steps were separated by SDS/PAGE, and proteins were stained with Coomassie Brilliant Blue G250. Ten microgram of protein was applied to each lane. M, prestained page ruler; lane 1, cell‐free extract; lane 2, cytoplasm; lane 3, pooled fractions from Q‐Sepharose; lane 4, pooled fractions from Phenyl‐Sepharose; lane 5, pooled fractions from Superdex 200.

Fig. 3

Pyruvate‐oxidizing activity of the purified PFOR1. Enzymatic activity was measured in 1.8‐mL anoxic cuvettes containing an overall liquid volume of 1 mL. The assay contained 5 μg PFOR, 200 μm CoA, and 50 μm TPP in buffer (50 mm Tris/HCl, 10 mm NaCl, 2 mm DTE, 4 μm resazurin, pH 7.5) under a 100% N₂ atmosphere at 66 °C. 30 μm Fd served as electron acceptor. The reaction was started by addition of 10 mm pyruvate. Reduction of Fd was measured at 430 nm.

Fig. 4

pH optimum and temperature profile of purified PFOR1. Temperature (A) or pH (B) dependence of the pyruvate‐dependent Fd reduction was measured in 1.8‐mL anoxic cuvettes containing an overall liquid volume of 1 mL under a 100% N₂ atmosphere at 20–80 °C (A) or 66 °C (B). The assay contained 1 mL of buffer A (50 mm Tris/HCl, 10 mm NaCl, 2 mm DTE, 4 µm resazurin, pH 7.5) or buffer B (50 mm Tris, 50 mm MES, 50 mm CHES, 50 mm CAPS, 50 mm Bis/Tris, 10 mm NaCl, 2 mm DTE, 4 µm Resazurin, pH 5–10), 5 μg PFOR, 200 μm CoA, 50 μm TPP, 30 μm Fd and 10 mm pyruvate. Shown is the average of two measurements from one representative experiment out of two independent replicates. Error bars represent the SEM.

Fig. 5

Cloning of pMU131_pfor1‐His. For the production of PFOR1‐His in Thermoanaerobacter kivui, the construct pMU131_pfor1‐His was cloned (A). Therefore, pMU131 backbone, including a S‐Layer promoter, was amplified using corresponding primers pMU131_for (1) and pMU131_rev (2) via PCR (size: 7192 bp) (B). Pfor1 was amplified from genomic DNA of T. kivui via PCR, using PFOR1‐His_for (3) and PFOR1‐His_rev (4) primers (size: 3598 bp) (C). PFOR1‐His_rev primer contained an additional DNA sequence coding for a 10x His‐tag. Amplified pfor1‐His and pMU131 backbone were fused via Gibson Assembly and transformed in E. coli HB101. Afterward, plasmids were isolated and digested with ScaI (D). The resulting sizes were 4241 bp and 3407 bp. M, Gene Ruler 1 kb DNA ladder.

Fig. 6

SDS/PAGE monitoring the purification process of PFOR1‐His. 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. M, prestained page ruler; lane 1, T. kivui cells; lane 2, cell‐free extract; lane 3, flow through; lane 4, wash fraction; lane 6, pooled Ni²⁺‐NTA elution fractions; lane 7, pooled size exclusion fractions.