Lactate dehydrogenases (LDHs) (EC 1.1.1.27) and Malate dehydrogenases (MalDHs) (EC 1.1.1.37) belong to a wide group of 2-ketoacid:NAD(P)-dependent dehydrogenases that catalyze the reversible conversion of 2-hydroxyacids to the corresponding 2-ketoacids (Holbrook et al., 1975). Both enzymes operate in central metabolism. LDHs achieve their function at the final stage of aerobic glycolysis and MalDHs are involved in the tri-carboxylic acid (TCA) cycle. They display the same protein fold (Rossmann fold) and a similar chemistry underlying their catalytic mechanism (Birktoft and Banaszak, 1983, Clarke et al., 1986, Hart et al., 1987a, Hart et al., 1987b, Clarke et al., 1988, Waldman et al., 1988). When the most chemically competent catalytic state is reached, LDH catalyzes the direct transfer of a hydride ion from the pro-R face of NADH to the C2 carbon of pyruvate (Pyr) to produce lactate; whereas MalDH converts oxaloacetate (OAA) into malate (Burgner and Ray, 1984, Fersht, 1985).

Numerous crystallographic structures of LDHs and MalDHs have been published (Iwata et al., 1994, Auerbach et al., 1988, Dalhus et al., 2002, Irimia et al., 2003, Coquelle et al., 2007, Arai et al., 2010, Coquelle et al., 2010 Dec 3, Ikehara et al., 2014, Kolappan et al., 2015, González et al., 2018, Roche et al., 2019, Iorio et al., 2021), allowing to describe their amino acids involved in NADH and substrate binding. For clarity, we refer to the numbering of amino acid accordingly to the one used for LDH (Eventoff et al., 1977). Pyruvate and oxaloacetate are oxoacids, which have a common negatively charged carboxylate extremity that is screened within the catalytic site by the positively charged lateral chain of R171, a universally conserved substrate-binding residue in all LDHs and MalDHs (Birkoft et al., 1982). When the most reactive conformational substate of the enzyme allowing the Michaelis complex formation is achieved, the mobile active-site loop moves down and close the catalytic site (Iwata et al., 1994, Coquelle et al., 2007). This phenomenon facilitates the catalytic site dehydration and a stronger anchoring of the substrate by hydrogen bonds with R109 (universally conserved in LDH and MalDH) and additional interactions induced by amino acid at position 102, Q for LDH and R for MalDH (Iwata et al., 1994, Coquelle et al., 2007). Canonical LDH structures with their ligands (HOLO states) revealed that the polar side chain of Q102, located on the mobile active-site loop, wrap the methyl of pyruvate allowing stabilizing this substrate in the appropriate orientation for catalysis within the catalytic site (Iwata et al., 1994). While in MalDHs, the lateral chain of R102 contributes to the screening of the second carboxylate extremities of OAA (Birkoft et al., 1982). The amino acid at position 102 is therefore considered as the most important substrate-discriminating residue between LDHs and MalDHs. Site-directed Mutagenesis experiments have demonstrated that the mutation Q102 to R on the mobile active-site loop transforms a LDH into a highly efficient MalDH (Wilks et al., 1988). The functional conversion from MalDH to LDH due the mutation of R102 to Q has been also attempted; however, this has been much less successful with respect to the catalytic efficiency that stayed quite low (Cendrin et al., 1993, Boernke et al., 1995, Katava et al., 2020). The reason for this lack of complete functional reversibility between these enzymes is very likely due to long-range epistatic effects. Epistasis is due to interactions between amino acid networks of a given protein that silent or enhance the consequence of a mutation depending on the presence or absence of other amino acids (Harms and Thornton, 2010, Olson et al., 2014, Miton et al., 2021).

Even if the LDH/MalDH super family is divided into two main functional groups, phylogenetic and biochemical studies have shown that it is actually populated by several subgroups that display specific signature sequences and different biochemical properties (Madern, 2002, Madern et al., 2004, Boucher et al., 2014, Roche et al., 2019, Katava et al., 2020, Brochier-Armanet and Madern, 2021, Iorio et al., 2022). Briefly, the super family contains the clades of MalDH 1 and 2 that are dimeric, a group of stricto sensu MalDH 3 mostly tetrameric, the stricto sensu LDH and a group whose sequence occupies an intermediate phylogenetic relationship between stricto sensu MalDH3 and LDH enzymes (Brochier-Armanet and Madern 2021).The intermediate group (IG) consists of several subgroups that reflect a large reservoir of sequences prone to evolve towards i) the functional conversion from MalDH to LDH and ii) allosteric regulation from non-allosteric MalDH-3 (Brochier-Armanet and Madern 2021). Note that most of the MalDHs from Archaea belong to the intermediate group.

Most of the bacterial LDHs are tetrameric enzymes, which exhibit a sigmoidal enzymatic profile with pyruvate, in the absence of fructose 1,6-bisphosphate (FBP), typical of homotropic allosteric activation of the reaction. When monitored in the presence of FBP, the enzymatic activity profile of allosteric LDHs display a hyperbolic profile demonstrating heterotropic allosteric activation (Arai et al., 2011).

With eukaryotic LDHs, the relationship to allostery is not yet resolved. Some works propose that they are non-allosteric enzymes (Pesce et al., 1967, Everse and Kaplan, 1973, LeVan and Goldberg, 1991, Holland et al., 1997), whereas others suggest they are reminiscent allosteric properties (Katava et al., 2017, Iacovino et al., 2022).

The protein folding and association pathway has been analyzed for the super family; it involves a series of sequential steps. In the first step, monomers in a molten globule state become more compact upon the formation of active dimeric species; in the second step the dimeric species condense to form active tetramers (Madern, 2000 and references therein). Abundant structural information has been obtained from the crystal structures of tetrameric LDHs and MalDH (Iwata et al., 1994, Auerbach et al., 1988, Dalhus et al., 2002, Coquelle et al., 2007, Coquelle et al., 2010 Dec 3, Ikehara et al., 2014, Kolappan et al., 2015, González et al., 2018, Roche et al., 2019, Iorio et al., 2021). Three molecular 2-fold axes named P, Q, and R (Rossmann et al., 1973) relate the tetramer subunits. LDHs have four active sites and two FBP-binding sites that are located at the AD and BC interfaces (AD-like interface) through the P-axis. The set of amino acid that participate to these sites, are absent in MalDHs type 3 and in MalDHs from Archaea, so that these enzymes are acknowledged as non-allosteric. In LDHs and MalDHs, the active site of each subunit lies near the interface along the Q-axis.

Studying present-day enzymes from the intermediate group is a relevant strategy to unravel the evolutionary steps that led to functional diversity and emergence of allosteric regulation within the LDH / MalDH super family. We consequently decided to investigate properties of an enzyme from Selenomonas ruminantium (S. rum) (Uniprot Q9EVR0), in which there is neither Q nor R at the substrate discriminating position 102, but an I. Even if this enzyme does not display a Q, it was characterized as a homotropically-activated LDH (Brochier-Armanet and Madern, 2021). In this follow-up study, the wild-type S. rum LDH crystal structure was solved. Its comparison with the structure from a canonical LDH that displays both homotropic and heterotropic activation reveals subtle local reorganization that explain why heterotropic activation by FBP is not possible. We enriched the characterization of the S. rum enzyme by a molecular dynamics simulation study. We have also analyzed the consequences of I to Q and I to R mutations at the substrate discriminating position 102, and solved the crystal structure of the former mutant. The present work offers new insights into the processes giving rise to the evolution of allostery in the super family of malate and lactate dehydrogenases.