Benoît Desguin

Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes.

A tethered niacin-derived pincer complex with a nickel-carbon bond in lactate racemase

Nickel pincers as enzyme cofactors Organometallic nickel complexes long synthesized in the laboratory exist naturally in enzymes as well. Desguin et al. determined the structure and metal-binding residues of the Ni-containing active site in bacterial lactate racemase (see the Perspective by Zamble). A dithiodinicotinic acid mononucleotide derivative cofactor binds Ni through sulfur and carbon bonds, resembling synthetic nickel pincer complexes. Genes encoding accessory proteins involved in the synthesis of this cofactor are widely distributed in other bacteria, suggesting its involvement in other enzymes. Science, this issue p. 66; see also p. 35 The bacterial interconversion of lactic acid isomers requires an enzyme with a nickel pincer complex. [Also see Perspective by Zamble] Lactic acid racemization is involved in lactate metabolism and cell wall assembly of many microorganisms. Lactate racemase (Lar) requires nickel, but the nickel-binding site and the role of three accessory proteins required for its activation remain enigmatic. We combined mass spectrometry and x-ray crystallography to show that Lar from Lactobacillus plantarum possesses an organometallic nickel-containing prosthetic group. A nicotinic acid mononucleotide derivative is tethered to Lys184 and forms a tridentate pincer complex that coordinates nickel through one metal-carbon and two metal-sulfur bonds, with His200 as another ligand. Although similar complexes have been previously synthesized, there was no prior evidence for the existence of pincer cofactors in enzymes. The wide distribution of the accessory proteins without Lar suggests that it may play a role in other enzymes.

Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria

MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)-glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.

Nickel-pincer cofactor biosynthesis involves LarB-catalyzed pyridinium carboxylation and LarE-dependent sacrificial sulfur insertion

Significance Nicotinic acid is a precursor of the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Previous studies revealed that nicotinic acid is required for lactate racemization, an enzymatic activity that enables microorganisms to produce or use both isomers of lactate. Lactate racemase (Lar) was shown to harbor a nicotinic acid-derived cofactor that coordinates a nickel ion, forming a pincer complex. The biosynthesis of this cofactor requires three accessory proteins: LarB, LarC, and LarE. Here, we describe this biosynthetic pathway by showing that LarB carboxylates and hydrolyzes the NAD precursor nicotinic acid adenine dinucleotide (NaAD) and LarE sacrificially inserts two sulfur atoms into the product of LarB. Finally, LarC inserts the nickel ion to form the mature cofactor. The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.

Enantioselective regulation of lactate racemization by LarR in Lactobacillus plantarum.

Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of l- and d-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the l-/d-lactate ratio and maximally induced in the presence of l-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarA induction by l-lactate. Gel retardation and footprinting experiments indicate that l-lactate has a positive effect on the binding and multimerization of LarR, while d-lactate antagonizes the positive effect of l-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.

Structural insights into the catalytic mechanism of a sacrificial sulfur insertase of the N-type ATP pyrophosphatase family, LarE

Significance Thiolation reactions are essential steps in the synthesis of numerous biological metabolites. To make the novel sulfur-containing cofactor of LarA, an Ni-dependent lactic acid racemase, LarE catalyzes a critical sulfur transfer reaction to a nicotinic acid-derived substrate by converting the proteins cysteine residue to dehydroalanine. In this study, crystal structures of ligand-free and several ligand-bound forms of LarE provide a structural basis for a catalytic mechanism that is further supported by structure-guided mutagenesis and functional assays. This work establishes LarE as a sulfur insertase within the N-type ATP pyrophosphatase family and presents a paradigm for sulfur transfer through sacrificing a catalytic cysteine residue. The lar operon in Lactobacillus plantarum encodes five Lar proteins (LarA/B/C/D/E) that collaboratively synthesize and incorporate a niacin-derived Ni-containing cofactor into LarA, an Ni-dependent lactate racemase. Previous studies have established that two molecules of LarE catalyze successive thiolation reactions by donating the sulfur atom of their exclusive cysteine residues to the substrate. However, the catalytic mechanism of this very unusual sulfur-sacrificing reaction remains elusive. In this work, we present the crystal structures of LarE in ligand-free and several ligand-bound forms, demonstrating that LarE is a member of the N-type ATP pyrophosphatase (PPase) family with a conserved N-terminal ATP PPase domain and a unique C-terminal domain harboring the putative catalytic site. Structural analysis, combined with structure-guided mutagenesis, leads us to propose a catalytic mechanism that establishes LarE as a paradigm for sulfur transfer through sacrificing its catalytic cysteine residue.

Unexpected complexity in the lactate racemization system of lactic acid bacteria

Analysis of lactate racemase (Lar) in lactic acid bacteria (LAB) has been a scientific challenge for many years, as indicated by the numerous contradictory reports on this activity. Recently, genetic and biochemical studies of the Lar system of Lactobacillus plantarum have unveiled the complexity of this particular enzymatic system. Lar activity is associated with LarA and its nickel-containing cofactor, synthesized from nicotinic acid adenine dinucleotide by the three biosynthetic enzymes: LarB, LarC, and LarE. In addition to these core Lar enzymes, a nickel transporter (Lar(MN)QO), a lactic acid channel (LarD) and a transcriptional regulator (LarR) which promotes expression of the lar genes in the presence of excess L-lactate are also part of the Lar system of Lb. plantarum and of many other LAB. These proteins promote racemization of external L-lactate, in addition to carrying out intracellular racemization. This additional outcome suggests that racemization of L-lactate is not only required for cell wall biosynthesis, as reported before, but may have additional roles in lactate production and utilization in LAB. Finally, bioinformatics analyses indicate that some Lar homologs probably catalyze reactions other than lactate racemization.

Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.

Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( kH/ kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.

Nickel–pincer nucleotide cofactor

A novel organometallic cofactor, nickel pyridinium-3,5-dithiocarboxylic acid mononucleotide, was recently discovered in lactate racemase (LarA) of Lactobacillus plantarum. This review summarizes the substantial progress made in uncovering the function of this cofactor as a transient hydride acceptor in the LarA mechanism. The latest developments related to cofactor biosynthesis reveal insights into a pathway in which LarB serves as a nicotinic acid adenine dinucleotide hydrolase/carboxylase, LarE acts as a sacrificial sulfur transferase, and LarC functions as a nickel insertase, forming the nickel-pincer nucleotide cofactor that becomes covalently tethered to LarA in some bacteria. Bioinformatic studies reveal a widespread occurrence of larA, larB, larC, and larE orthologs in microorganisms, and additional roles for the cofactor are considered.

Lactate Racemase and Its Niacin-Derived, Covalently-Tethered, Nickel Cofactor

The biological racemization of d- and l-lactic acid was first reported in 1936 and has remained mysterious for eight decades. Recently, considerable advances have been achieved by studying the lactate racemase system in Lactobacillus plantarum. In this species, two operons encompassing a total of nine genes are responsible for this activity, with four genes being strictly required. LarA was shown to be the lactate racemase harboring a nickel ion coordinated by a niacin-derived (SCS) pincer complex. A likely mechanistic hypothesis is that the pincer complex reversibly captures a hydride from lactate, forming the achiral pyruvate intermediate. The three accessory proteins required for lactate racemization catalyze carboxylation, sulfur insertion, and nickel incorporation steps during the synthesis of the lactate racemase cofactor from nicotinic acid adenine dinucleotide. LarE, when expressed in the presence of the other two other accessory proteins LarB and LarC, is able to activate the LarA apoprotein in vitro. This suggests the mature cofactor is assembled on LarE before its transfer to the LarA apoprotein. The presence of the lactate racemase accessory proteins in many genomes lacking larA suggests a wider use of the cofactor in the prokaryotic world.