Crystal structure and biochemical characterization of malate dehydrogenase from Metallosphaera sedula

Highlights

• Crystal structure of the archaeal malate dehydrogenase from Metallosphaera sedula (MsMDH) was determined.

• Dual cofactor specificity of MsMDH was elucidated through the structural insight.

• The MsMDH structure in complex with malate showed a conformational change at the active site upon the substrate binding.

Abstract

Metallosphaera sedula is a thermoacidophilic autotrophic archaeon and known to utilize the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) as a carbon fixation pathway. The 3-HP/4-HB cycle in M. sedula is associated with central metabolism, and malate dehydrogenase (MDH) is an enzyme involved in the central metabolism that converts malate to oxaloacetate. To elucidate the enzymatic properties of MDH from M. sedula (MsMDH), we determined the crystal structure of MsMDH as a complex with NAD⁺ and a ternary complex with malate and NAD⁺. Based on its complex structures and biochemical experiments, we observed that MsMDH can utilize both NAD⁺ and NADP⁺ as a cofactor. In addition, we revealed that MsMDH shows a conformational change at the active site upon substrate binding. Based on the comparison with other MDHs, we revealed that MsMDH was distinguished from general MDHs due to a Lys80 residue, and this difference is likely to influence the unique cofactor specificity of MsMDH.

Introduction

In prokaryotes, six distinct autotrophic CO₂ fixation pathways have been identified so far. Of these pathways, 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle is one of the recently discovered pathways [1]. The 3-HP/4-HB cycle was first identified in Metallosphaera sedula strain [1], and M. sedula has since emerged as a significant archaea in the CO₂ fixation pathway. According to sequence information, M. sedula belongs to the order Sulfolobales, which belongs to the thermoacidophilic autotrophic phylum Crenarchaeota [[2], [3], [4], [5]]. M. sedula was first isolated from the volcanic area of Pisciarelli Solfatara in Naples, Italy [6], and has optimal growth conditions of 75 °C and pH 2.0 [6,7].

M. sedula is known to utilize the 3-HP/4-HB cycle [8,9], which is roughly divided into two sub-pathways [1]. In the first sub-pathway, two bicarbonate molecules are added to an acetyl-Coenzyme A (CoA), and succinyl-CoA is formed as an intermediate. The second sub-pathway is the regeneration of two acetyl-CoA molecules from succinyl-CoA. The 3-HP/4-HB cycle of M. sedula is associated with central metabolism by supplying succinyl-CoA to activate the tricarboxylic acid (TCA) cycle [1,9,10]. Succinyl-CoA is converted to succinate by succinyl-CoA synthetase, and succinate is then converted to malate by succinate dehydrogenase and fumarate hydratase. Finally, malate dehydrogenase (MDH) is involved in the dehydrogenation reaction of malate to oxaloacetate (OAA). In a variety of organisms including M. sedula, the OAA formed by MDH is utilized as a crucial precursor in many important metabolic pathways: the TCA cycle, glyoxylate bypass, amino acid synthesis, and gluconeogenesis [11,12].

MDH enzymes from a diversity of organisms have been extensively studied. MDH enzymes catalyze an oxidation reaction that converts malate to OAA, and the reverse reaction also occurs [11]. This catalytic reaction is accompanied by the reduction of NAD⁺ to generate NADH. Based on phylogenetic analysis, MDHs are known to be distinguished into two main phylogenetic groups: general MDHs and lactate dehydrogenase (LDH)-like MDHs [13,14]. The dimeric structure is a common form of general MDHs, and dimeric MDHs are divided into NAD⁺-dependent MDHs and NADP⁺-dependent MDHs depending on the cofactor specificity. By contrast, the LDH-like MDHs have a different characteristic from general MDHs. Unusually, LDH-like MDHs are mostly composed of a tetrameric structure and their amino sequences are more similar to LDH than MDH [13]. That is, these enzymes show the enzymatic activity of MDH in the form of LDH.

Although MDH enzymes have been studied extensively, structural studies on LDH-like MDHs are insufficient compared to studies of general MDHs. Additionally, M. sedula is an important archaeon for carbon fixation, but the study on MDH from M. sedula (MsMDH) has not yet been reported. In this study, we determined the crystal structure of MsMDH as a complex with NAD⁺ and as a ternary complex with malate and NAD⁺. Interestingly, we revealed that MsMDH has the unique feature such as dual cofactor specificity and observed that a conformational change occurs at the active site of MsMDH upon substrate binding.