Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase

Abstract

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.

FIG 1

The relative activity of recombinant Cthe_0344 (MalE) (■) and Cthe_0345 (MDH) (▲) at various pH values with standard assays conditions at 50°C (MalE) and 25°C (MDH).

FIG 2

Thermostability profile of Cthe_0345(MDH) at 4°C (▲), 25°C (◆), and 60°C (■) (A) and Cthe_0344 (MalE) at 37°C (●) and 60°C (■) (B) under standard assay conditions at pH 7.0 with 20 mM NH₄⁺and 5 mM Mn²⁺.

FIG 3

Relative activities of recombinant Cthe_0344 (MalE) at pH 7.0 in the presence of various concentrations of NH₄Cl with 5 mM Mn²⁺ (A) and various concentrations of PP_i with 20 mM NH₄⁺ and 5 mM Mn²⁺ (B).

FIG 4

Molecular phylogenetic analysis of Cthe_0345 (MDH) by the maximum likelihood method. The gray box indicates a grouping of putative LDH coding genes. The black box indicates a grouping of MDH genes which have a neighboring MalE gene. Arrows indicate sequences for which corresponding enzymes have been purified and characterized. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

FIG 5

Molecular phylogenetic analysis of Cthe_0344 (MalE) by the maximum likelihood method. The box indicates the group of MalE with a neighboring MDH gene. Arrows indicate sequences for which corresponding enzymes have been purified and characterized. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

FIG 6

Amino acid alignment of Cthe_0345 (MDH) and Cthe_1053 (LDH) from C. thermocellum and b3236 (MDH) from E. coli K-12 MG1655. Shaded areas indicate positions 18, 86, 90, 212, and 216 based on the amino acid positions of Cthe_0345 (MDH).

FIG 7

Putative pyruvate-producing pathways in C. thermocellum under normal intracellular conditions (A), with high intracellular PP_i and NH₄⁺ (B), with low intracellular PP_i and high NH₄⁺ (C), and with low PP_i and NH₄⁺ (D).