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 Lys(184) and forms a tridentate pincer complex that coordinates nickel through one metal-carbon and two metal-sulfur bonds, with His(200) 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.

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.

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.actic acid, composed of both L-and D-isomers, is a widespread organic compound produced during microbial fermentations via stereospecific lactate dehydrogenases. Certain bacteria possess the ability to interconvert the two enantiomers by using lactate racemase, which was only recently described in genetic (1), structural (2), synthetic modeling (3), and computational studies (4, 5).LarA from Lactobacillus plantarum is responsible for lactate racemase activity. This Ni-dependent enzyme (1) contains a newly identified cofactor, pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), that is covalently attached to an active-site lysine residue (2). Most interestingly, P2TMN binds an Ni atom using sulfur, carbon, and sulfur atoms of an SCS-pincer complex (2), making LarA the ninth discovered Ni-dependent enzyme (6).Synthesis of Ni-bound P2TMN occurs through a pathway involving three proteins encoded in the lar operon (i.e., LarB, LarC, LarE) (1). LarB is a carboxylase/hydrolase that produces pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN) from nicotinic acid adenine dinucleotide (7). LarE then converts P2CMN into P2TMN through two successive sulfur transfer reactions. Finally LarC is thought to provide the Ni atom to generate the active form of the pincer cofactor of LarA (7) (Fig. 1).The amino acid sequence of LarE suggests it has two domains. The N-terminal domain is homologous to N-type ATP pyrophosphatase (PPase) domains (8), containing a conserved SGGxDS motif that binds and hydrolyzes ATP to form AMP and pyrophosphate. Examples of enzymes with this domain are guanine monophosphate (GMP) synthetase, nicotinamide mononucleotide (NMN) synthetase, and nicotinamide adenine dinucleotide (NAD) synthetase. These enzymes activate substrate carboxyl or carbonyl groups by adenylylation (AMPylation) (9). The C-terminal domain of LarE has no homology to any member of the N-type ATP PPase family, which use their C-terminal domains to recognize specific substrates and catalyze their versatile reactions. We theref...

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 singleturnover 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.lactate | racemase | nicotinic acid dinucleotide | sacrificial enzyme