Mini Review - (2018) Volume 9, Issue 2

Lactate Racemization and Beyond

Benoit Desguin*
Institute of Life Sciences, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium
*Corresponding Author: Benoit Desguin, Institute of Life Sciences, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium Email:


Enzymatic racemization of lactate has been reported in several bacterial species, including Lactobacillus species. The role of lactate racemase (Lar) is still a matter of debate and is probably dependent if the species in which it is found is a lactate producer, a lactate consumer, or both. A transcriptomic experiment revealed the involvement of two operons of 9 genes in lactate racemization in L. plantarum: the larR (MN) QO and the larABCDE operons. The lactate racemase, LarA, has been shown to harbour a tethered nickel pincer complex, which we call Nickel Pincer Nucleotide or NPN in this review. This cofactor seems well adapted to catalyse lactate racemisation by a hydride transfer mechanism. The cofactor is synthesized from nicotinic acid adenine dinucleotide by the NPN biosynthetic enzymes: LarB, LarC, and LarE. LarD is an aquaglyceroporin, LarR a transcriptional regulator, and Lar (MN) QO a three-component nickel transporter. Lactate racemase gene was reported to be widespread in bacterial and archaeal genomes. We suggest that many other enzymatic functions are present in the LarA superfamily of enzymes in addition to lactate racemization.

Keywords: Lactate racemase; Nickel; Pincer complex; Lactobacillus plantarum; Aquaglyceroporin; Transcriptional regulator; Cofactor

The Lactate Racemization Operon

Enzymatic racemization of lactate was first reported in 1936 in Clostridium beijerinckii (formerly C. butylicum ) [1] and was then detected in Staphylococcus aureus (formerly S. ureae ), in Lactobacillus sakei (formerly L. sake ) [2], in L. plantarum [3], and in several other Lactobacillus sp. [4], as well as in the rumen bacteria Selenomonas ruminantium and Megasphaera elsdenii [5,6] and in halophilic archaea [7].

The role of lactate racemase (Lar) is still a matter of debate and is probably dependent on the species in which it is found. In bacteria consuming lactate, like M. elsdenii , Lar enables the consumption of both L- and D-lactate isomers, despite the presence of only one D- or L-lactate dehydrogenase [6]. In lactic acid bacteria, the presence of a lactate racemase is less straightforward, as lactate is a waste product of fermentation and its racemization is not required for growth. In L. plantarum , Lar was suggested to be used as a rescue pathway for the formation of D-lactate, which is incorporated in the cell wall and confers to the bacteria a resistance to the vancomycin antibiotic [8,9]. This role can be extended to the growth conditions in which the bacteria perform malolactic fermentation, producing only L-lactate [10]. However, the presence of the lactic acid channel LarD suggests that this is probably not the only role Lar fulfils in lactic acid bacteria. Lactate racemization of externally produced L-lactate in D-lactate consuming bacteria could be a way for the lactate racemizing bacteria to generate a proton gradient across its cell wall, if a hypothetical transporter of L-lactate was present in these species [10].

The identity of the Lar enzyme remained unknown until a transcriptomic experiment performed in 2014 revealed the involvement of two operons of 9 genes in lactate racemization in L. plantarum [9,11]. These genes were shown to encode a nickel transport system Lar(MN)QO, a nickel-dependent lactate racemase LarA, a set of three proteins of unknown function LarB, LarC and LarE necessary for the maturation of the racemase, a lactic acid channel LarD [12], and a transcriptional regulator LarR responding to the enantiomeric excess of L-lactate [13]. These proteins constitute the lactate racemase system found in L. plantarum and in many other Lactobacillus species (Figure 1) [10].


Figure 1: The Lar system of L. plantarum .

An Intriguing Cofactor in Lactate Racemase

In 2015, LarA was shown to contain a covalently tethered (SCS) Ni (II) pincer complex, which catalyses lactate racemization (Figure 2) [14]. Pincer complexes are well known in organometallic chemistry but have never been observed in any wild type enzyme before. For simplicity, we call it the nickel pincer nucleotide or NPN. This cofactor exhibits many original features never previously reported in any biological molecule, such as a stable nickel-carbon bond and the presence of a unique pincer structure made of a pyridinium ring substituted by two sulfur-containing groups (Figure 2). Both the mechanism of action of NPN in this reaction and the reason why a nickel ion is required are still unknown.


Figure 2: A. LarA structure, B. Catalytic site with NPN, C. Proposed catalytic mechanism.

Past reports suggested that there is a hydride transfer mechanism for lactate racemisation [15,16]. In light of the new cofactor structure, the existence of such a mechanism seems plausible since NPN appears well adapted for hydride transfer chemistry that would generate a pyruvate/NPNH intermediate. This hypothesis has been confirmed by recent DFT calculations, which suggest that the role of NPN is either to destabilize the intermediate [17] or to provide greater hydride-addition reactivity [18]. Following the discovery of NPN in lactate racemase, another mechanism based on proton coupled single electron transfer (PCET) and cleavage of the C-C bond between the alpha carbon and the carboxylic group was proposed based on quantum mechanics/ molecular mechanics calculations [19]. In this case, a paramagnetic cofactor, with Ni(III), is predicted to be present in the ground state of the enzyme, and a single acidic-basic residue (His108) is predicted to perform the deprotonation of both lactate isomers, with the reaction then proceeding through different tightly bound intermediates, i.e. acetaldehyde and carbon dioxide radical anion (CO2 -). Even though this hypothetic mechanism is plausible, we consider that it is very unlikely since it is not compatible with old kinetic isotope effect (KIE) experiments that indicated a primary isotope effect for lactate racemization in C. beijerinckii [16], meaning that the C-H bond is cleaved during catalysis, and it is very likely that the C. beijerinckii racemase is a member of the LarA family. Interestingly, in 2016, a nickel pincer complex featuring some similarities with NPN was synthesized and shown to mediate dehydrogenation of alcohols [20].

Shortly after the discovery of NPN, its biosynthesis was shown to involve LarB, LarC and LarE and nicotinic acid adenine dinucleotide (NaAD) as a precursor [21]. LarB starts by catalysing the carboxylation of the nicotinic ring that is accompanied by hydrolysis of the phosphoanhydride bond, forming pyridinium biscarboxylic acid mononucleotide (P2CMN). The energy generated by the hydrolysis of the phosphoanhydride bond probably facilitates the carboxylation reaction, although this coupling remains mysterious. LarE converts both the carboxylate groups of P2CMN into thiocarboxylate groups by an ATP-dependent sacrificial sulphur insertion, forming pyridinium bisthiocarboxylic acid mononucleotide (P2TMN). As the two sulphur atoms inserted into P2CMN are derived from one cysteine of LarE, two LarE proteins are required for the synthesis of one P2TMN [22]. Finally, nickel-containing LarC catalyses the nickel insertion between the two sulphur atoms and the pyridinium carbon 4 of P2TMN,generating NPN [21]. Finally, in case of covalent NPNylation, NPN reacts with a lysine of LarA, forming a thioamide bond (Figure 3).


Figure 3: The Ni-pincer nucleotide and its biosynthesis [21]. Starting from nicotinic acid adenine dinucleotide (NaAD), LarB carboxylates the pyridinium ring and hydrolyses the phosphoanhydride, forming pyridinium biscarboxylic acid mononucleotide (P2CMN). In an ATPdependent reaction, two LarE molecules desulfurise themselves converting a cysteine residue into a dehydroalanine while the sulphur atoms are incorporated into P2CMN, forming pyridinium bisthiocarboxylic acid mononucleotide (P2TMN). LarC then incorporates the nickel ion and the NPN cofactor is covalently attached to the racemase through a thioamide bond.

Beyond Lactate Racemisation

A bioinformatic analysis of over 1000 prokaryotic genomes revealed the presence of the genes coding for NPN-biosynthetic enzymes and LarA homologs in 10% of the studied genomes [11] suggesting a wide distribution of lactate racemization in the prokaryotic world. Nevertheless, very few organisms have been shown to actually racemize lactate [1-7]. Furthermore, it has been hypothesized that some LarA homologs likely do not racemize lactate but catalyze some other, probably similar, reaction [10]. LarA is indeed part of a large superfamily, the superfamily of unknown enzymes DUF2088 (or PF09861), which comprises 1126 identified sequences in 662 species [23]. The majority of members of this superfamily are found in bacteria, mostly in Firmicutes (270 species), in Actinobacteria (74) and in Proteobacteria (83 species), yet some members have been found in Archaea (63 species) and even in Eucaryota (17 species, mostly green algae and oomycetes). In this diversity of members, the lactate racemase compose only a fraction of the sequences, 1 in 6 approximately, the vast majority of the members of this family code for enzymes of unknown function (Figure 4). As these enzymes are homologs of LarA, they probably also catalyse epimerization or racemization reactions, which in both cases involve the inversion of a chiral center. Nevertheless, other types of isomerization could also be catalysed by hydride transfer and may also be considered, like cis-trans isomerization or intramolecular oxidoreductions. The observation that some species harbor several LarA paralogs, e.g. 4 paralogs for M. elsdenii (Figure 4), strengthens the hypothesis that several other reactions are catalyzed by the members of this superfamily.


Figure 4: Alignment of 1126 members of the DUF2088 superfamily with the members present in L. plantarum (1 member), L. sakei (1 member), C. acetobutylicum (1 member), C. beijerinckii (2 members), and M. elsdenii (4 members) indicated. The alignment was downloaded from the European Bioinformatic Institute (EMBL-EBI) and visualized with dendroscope [24].

Furthermore, the genes coding for NPN-biosynthesis enzymes were also identified in another 15% of the studied genomes that lack larA [11], suggesting that enzymes that are not homologous to LarA use NPN as well. These enzymes could catalyse many other reactions requiring NPN, not necessarily involving an epimerization or racemization reaction and not necessarily involving a hydride transfer mechanism.


As a conclusion, we can say the lactate racemization is probably just the tip of the iceberg of all NPN-dependent enzymatic reactions there is still a lot to discover on the subject.


Citation: Desguin B (2018) Lactate Racemization and Beyond. J Bacteriol Parasitol 9: 335.

Copyright: © 2018 Desguin B. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.