Malate dehydrogenase (MDH) is a ubiquitous enzyme involved in cellular respiration across all domains of life. MDH’s ubiquity allows it to act as an excellent model for considering the history of life and how the rise of aerobic respiration and eukaryogenesis influenced this evolutionary process. Here, we present the diversity of the MDH family of enzymes across bacteria, archaea, and eukarya, the relationship between MDH and lactate dehydrogenase (LDH) in the formation of a protein superfamily, and the connections between MDH and endosymbiosis in the formation of mitochondria and chloroplasts. The development of novel and powerful DNA sequencing techniques has challenged some of the conventional wisdom underlying MDH evolution and suggests a history dominated by gene duplication, horizontal gene transfer, and cryptic endosymbiosis events and adaptation to a diverse range of environments across all domains of life over evolutionary time. The data also suggest a superfamily of proteins that do not share high levels of sequential similarity but yet retain strong conservation of core function via key amino acid residues and secondary structural components. As DNA sequencing and ‘big data’ analysis techniques continue to improve in the life sciences, it is likely that the story of MDH will continue to refine as more examples of superfamily diversity are recovered from nature and analyzed.

Malate dehydrogenase (MDH) is an enzyme that plays critical roles in the aerobic respiration process that is central to the majority of life on Earth. Specifically, MDH catalyzes the interconversion between malate and oxaloacetate via the reduction of NAD+ to NADH as a part of the tricarboxylic acid (TCA) cycle that takes place within the mitochondria of eukaryotes [1]. MDH is also a critical component of the malate-aspartate shuttle that is responsible for transporting electrons produced during glycolysis in the cytoplasm to the mitochondrial matrix, where the oxidative phosphorylation process takes place [2]. These distinct yet related functions for MDH are the result of a rich evolutionary history for this enzyme in which a diverse array of MDH and MDH-like proteins have arisen to allow organisms across the spectrum of life to adapt to a host of environmental challenges. The ubiquity of MDH across living organisms and the key conserved roles that it plays in the maintenance of life makes it an ideal model for the study of molecular evolution as well as the consideration of some of the most profound unanswered questions about the development of cellular complexity. The development of the sophisticated cellular physiology commonplace among modern eukaryotes is largely the result of two critical events in the history of Earth: the rise of aerobic respiration and the endosymbiotic events that gave rise to mitochondria and chloroplasts as proverbial ‘powerhouses’ of the cell [3–7]. Given MDH’s intimate associations with aerobic respiration, it is tempting to attribute these two events as essential for the development of the diverse array of MDH family members present across the diversity of life on Earth. Indeed, many studies have reached the conclusion that the similarities that exist between different isoforms of MDH in humans and other multicellular eukaryotes support an endosymbiotic origin for this enzyme amongst these organisms [8–13]. However, it is also the case that MDH family members can be found in a plethora of bacteria and archaea that are anaerobic in nature, where they help to protect organisms against free radical formation as well as produce succinic acid as part of a reductive form of the TCA cycle [8,14,15]. Interestingly, recent studies utilizing modern high-throughput sequencing techniques suggest that previous theories that have established endosymbiosis as the key action responsible for MDH transfer between different organisms may require revisitation and reconsideration [16]. This essay seeks to summarize current understandings of MDH evolution and establish what questions need next to be answered to come to a more complete understanding of the complex story of this crucial enzyme.

The story of MDH is likely a story of gene duplication and horizontal gene transfer events that have allowed for this family of enzymes to develop a diverse set of ways to support the respiration process in cells in parallel with the evolution and increasing complexity of the respiration process itself. Over the course of millions of years, the MDH family has developed clades largely based on structural rather than sequence similarity. Recent sequencing work has organized MDH across all three domains of life (bacteria, archaea, eukarya) into three primary groups: a dimeric group that primarily exists in bacteria, a dimeric group that primarily exists in eukaryotes, and a tetrameric group that is the primary source of MDH in archaea but which is scattered amongst all three domains. This third subgroup includes not only MDH enzymes but also L-2-hydroxyisocaproate dehydrogenases (HicDHs) and enzymes that bear resemblance to the enzyme lactate dehydrogenase (LDH) (discussed in more detail below) [16]. In mammals, two main forms of MDH exist that can be distinguished by the compartments in which they operate: one isoform restricts itself to the cytoplasm and participates in the malate/aspartate shuttle while the other isoform is located within mitochondria to engage with the TCA cycle [13]. What these isoenzymes have in common is that both are encoded within the nucleus and synthesized in the cytosol, like other nucleus-encoded proteins. However, the cytoplasmic isoform of MDH remains in the cytosol after being synthesized while the mitochondrial isoform is imported into the mitochondrial matrix as the result of an extension of N-terminal residues lacking in cytoplasmic MDH which targets this isoform to the outer mitochondrial membrane [17,18]. This extension acts as a signal sequence to allow the isoenzyme to enter the mitochondria, a process which is dependent on the use of precursor proteins and an electrochemical gradient across the outer mitochondria membrane [19].

A sequence comparison of the two MDH isoforms within humans shows that there is only ∼20% sequence identity between them, a fact that suggests either an evolutionarily-distant gene duplication event or two distinct horizontal transfers as the potential origin story for these isoforms. However, a comparison of the two isoforms on strictly structural terms [20] suggests a high degree of similarity of secondary and tertiary structure between them despite a disparity in sequence (Figure 1), revealing the critical preservation of key residues and, thus, overall protein shape as critical for the preservation of overall MDH function amongst family members despite sequence dissimilarity. Nonetheless, this paradox of similar function despite great sequential disparity suggests that the evolutionary history of the two human MDH isoforms may be more complex that originally thought and that they may in fact have quite distinct lineages from each other [16]. This is supported by the fact that when comparing the human isoforms to those in other eukaryotes, the similarity of the same isoform between different organisms is greater than the similarity between the different isoforms in the same cell [12].

The isoforms of MDH are most conserved in their active site regions, representing how the function of the enzyme is needed for many metabolic pathways. The mechanism involves what is known as a ‘hydrophobic vacuole’ [9,11–13], where an external loop of the enzyme uses the hydrophobic external loop of the enzyme to create a vacuole for the substrate (Figure 2). The ‘vacuole’ contains binding sites for both the enzyme and substrate and utilizes a catalytic triad of residues to perform its function. Conformational change induced by oxaloacetate binding leads to the external loop to close the ‘vacuole’. Not only is the process similar in all MDH isoforms, but the affinity between enzyme, substrate, and cofactor are also conserved, by the conservation of catalytic triad residues that are needed for the binding process.

Amongst eukaryotes, several additional compartment-confined isoforms of MDH can be found within specific groups of organisms. For instance, the budding yeast Saccharomyces cerevisiae has a third MDH isoform in addition to the two described above that localizes to peroxisomes [21,22]. In plants, a third major dimeric MDH isoenzyme exists that utilizes NADP+ instead of NAD+ as a cofactor [23–25]. Inside the stroma of the chloroplast, this MDH isoenzyme contributes to the dicarboxylic acid cycle of photosynthesis to produce sucrose via the conversion of oxaloacetate to malate and the oxidation of NADPH to NADP+ (the opposing mechanism of that utilized by mitochondrial MDH). Plants are also home to additional glyoxysomal and peroxisomal isoforms as well [23–25].

As previously mentioned, recent high-throughput sequencing work has revealed a diverse clade of related MDH isoforms that exist in tetrameric form instead of the dimeric form found in most bacteria and eukaryotic cells. This tetrameric version is all the more of interest when studying the evolution of MDH when one notes the strong similarities that exist between the structure of these MDH enzymes with LDH, a tetrameric protein primarily responsible for a reversible reaction that converts pyruvate to lactate utilizing NADH as a coenzyme [26]. Both LDH and MDH catalyze a reversible reaction between 2-hydroxyacids and 2-ketoacids, and the tetrameric form of MDH, like LDH, is the primary form found in archaea but can be found across all three domains of life, albeit rarely amongst eukaryotes. This apparent connection between the MDH and LDH has been studied extensively [8–13,16,26] and has led to the development of some intriguing theories to attempt to explain their relationship. Because these enzymes are part of a central metabolic pathway, any evolutionary trajectory would likely have been stepwise and lead to new functionality gradually, thereby leaving a trail that could be detected through extensive comparative sequence and structural analysis.

An intriguing discovery within the hyperthermophilic archaea Ignicoccus islandicus has led to novel and exciting ideas about how LDH may have evolved from pre-existing MDH. In I. islandicus, the MDH isoform recognizes oxaolacetate, the primary substrate of MDH, as its primary substrate but also shows low levels of affinity for pyruvate, the primary substrate for LDH, as well. This finding led to the discovery of additional archaeal examples of MDH isozymes possessing dual MDH/LDH substrate activity and the theorizing that LDH evolved as the result of a gene duplication event from tetrameric MDH [27]. The precise evolutionary link seems to reside within the gradual change in members of a catalytic triad that has remained structurally conserved throughout the MDH/LDH superfamily despite significant sequential changes across the remainder of the proteins [16]. The possession of both MDH and LDH would permit a cell to utilize both anaerobic and aerobic conditions for respiration and thus possess a selective advantage that promoted the widespread presence of both enzymes across all domains of life. Thus, the MDH/LDH superfamily may be defined in part by both a conformational diversity (dimeric versus tetrameric structure) and functional promiscuity (oxaloacetate versus pyruvate substrate) that has led to its strong adaptability across evolutionary time in a ubiquitous fashion.

One of the most critical events in cellular evolutionary history is the endosymbiosis between an ancestral archaeal cell and an alphaproteobacterium [3] resulting in a mutualistic relationship that eventually led to a series of horizontal gene transfers from the alphaproteobacterium to the archaeal cell and the development of a co-dependent relationship between the two entities that is now recognized as eukaryotic cells with mitochondria. A separate endosymbiotic event involving a cyanobacterial cell resulted in the development of eukaryotic cells with chloroplasts. Ancestrally, endosymbionts and their hosts represented distinct domains of life in which their amalgamation generated entirely new combinations of biochemical capabilities and allowed the two intertwined species to thrive in environments that would be inhospitable to either alone [28]. Given the role of these endosymbionts in the production of energy within eukaryotic cells and the specific role played by MDH in the maintenance of aerobic respiration, it would naturally follow that MDH arrived in eukaryotes via horizontal gene transfer from the endosymbionts followed by gene duplication events creating the different isoforms of MDH found in modern eukaryotes. Indeed, this theory of an endosymbiotic origin for eukaryotic MDH has persisted in the literature across many decades [8–13] and was supported by comparative sequential data across several key model organisms. However, access to larger-scale sets of sequence data for MDH across all domains of life has led to the challenging of this theory. Specifically, an examination of dimeric MDH isozymes across bacteria and eukaryotes does not reveal any significant presence of sequences with similarity to alphaproteobacteria, which would eliminate this bacterial class as the provider of MDH to eukaryotes and, therefore, alphaproteobacterial ancestors to mitochondria as the donors of MDH to what became the eukaryotic nuclear genome. There is substantial evidence for modern mitochondria having an alphaproteobacterial ancestry [29–31], eliminating the potential alternative explanation for the endosymbiotic theory of MDH transfer and evolution. Collectively, this data suggests that if endosymbiosis was indeed responsible for the presence of eukaryotic MDH, then it would have had to have been from a cryptic endosymbiosis event involving a gammaproteobacterium which is no longer present in eukaryotes as an endosymbiont. Similarities between eukaryotic and contemporary gammaproteobacterial MDH add further credence to this idea [16]. Most likely, however, a horizontal gene transfer event of unknown origin to an ancestral archaeal cell resulted in the initial presence of MDH in what are now eukaryotes. In this model, the differences between eukaryotic MDH isozymes are explained by a common ancestral MDH gene that may have been duplicated before the invasion of primordial eukaryotes by bacteria to produce mitochondria [13]. Thus, presence of dimeric MDHs in eukaryotes is better explained by cryptic endosymbiosis, lateral gene transfer, or the presence of a pre-existing gene in a proto-eukaryotic ancestor [8]. These findings are exciting in the sense that new and more efficient DNA sequencing techniques have paved the way for increased capacity to analyze larger data sets and, in turn, uncover previously unknown patterns that challenge preconceived notions of evolution and improve our overall understanding of the development of complex life forms.

When all of the above information is combined, the history of MDH can be seen as a history that has existed in parallel with the key events in the history of life leading to eukaryotic organisms (Figure 3). While MDH appears to have ties to both the rise of aerobic respiration and the entering of proteobacteria into endosymbiotic relationships with ancestral archaea, it is also evident that MDH’s ancestry has evolved to accommodate and exploit these events. Taken together, the data suggests that the initial split from MDH’s common ancestor gave rise to what became the dimeric MDHs found in eukaryotes and bacteria and the tetrameric MDHs found primarily in archaea. Within the latter group, further evolutionary events led to the rise of LDH-like MDH enzymes in bacteria with substrate promiscuity between oxaloacetate and pyruvate as well as canonical LDHs. Horizontal gene transfer, in turn, spread LDH to eukaryotes and some of the archaeal species [26]. When this proposed phylogenetic tree is viewed against the significant episodes of life history, MDH can be surmised to have initially developed in an anaerobic environment. As photosynthesis within cyanobacteria began the process of oxygenating the Earth’s atmosphere, mutations to MDH allowed it to play a key role in the rise of exploiting this new abundant resource for aerobic respiration. Likely gene duplication events and horizontal gene transfer events (potentially via cryptic endosymbiosis) paved the way for the development of multiple isozymes of MDH that could further enhance aerobic respiration in these cells and further bolster the adaptive advantages these cells would have in an oxygenated environment. Over time, the endosymbiotic events leading to mitochondria and chloroplast formation greatly enhanced the efficiency of what became eukaryotic aerobic respiration; however, the pre-existing MDHs in ancestral archaeal cells were sufficient for the roles to be played by MDH in the emergent eukaryotic cell. Thus, whatever copies of MDH arrived in the eukaryote via endosymbiosis became lost over time but before significant divergence amongst eukaryotic species.

Given what new advances in DNA sequencing technology and ‘big data’ analysis have yielded with respect to our understanding of MDH evolution, it would be foolish to suggest that what has been presented here is the definitive end to this story. Indeed, one of the powers of this increased capacity for large dataset analysis has been the consideration of larger classes of bacterial and archaeal specimens that are either being newly discovered or have been poorly understood. The continuing enrichment of the MDH sequence dataset with samples like the I. islandicus substrate-promiscuous LDH-like MDH will continue to reveal new exceptions that may continue to alter our fundamental understanding of MDH’s history. Such new data may also provide novel insights on the roles that MDH may have had in the development of aerobic respiration and eukaryotic life on Earth.

  • Malate dehydrogenase has evolved into distinct clades across bacteria, archaea, and eukarya over millions of years yet have maintained a strongly conserved core function for the conversion of oxaloacetate to malate utilizing NADH as a cofactor.

  • Gene duplication, horizontal gene transfer, and cryptic endosymbiosis may have contributed to MDH’s ability to be a critical roleplayer in the rise of aerobic respiration across all domains of life, the development of eukaryotic cells via formation of mitochondria and chloroplasts, and the evolution of LDH as an additional key player in the modern cellular respiratory process.

  • Novel high-throughput DNA sequencing techniques have challenged conventional ideas that MDH was introduced to eukaryotic cells by their mitochondrial and chloroplastic proteobacterial ancestors.

The authors declare that there are no competing interests associated with the manuscript.

All authors took equal roles in the conception, research, and writing of this manuscript.

The authors are grateful to Dr. Joseph Provost of the University of San Diego and the entire Malate Dehydrogenase CUREs Community for the opportunity to write this manuscript.

HicDH

L-2-hydroxyisocaproate dehydrogenase

LDH

lactate dehydrogenase

MDH

malate dehydrogenase

TCA

tricarboxylic acid

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