The Nickel‐Pincer Complex in Lactate Racemase Is an Electron Relay and Sink that acts through Proton‐Coupled Electron Transfer

Wang, Binju; Shaik, Sason

doi:10.1002/anie.201612065

Cited by 17 publications

(10 citation statements)

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“…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).…”

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confidence: 99%

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

Fellner

Desguin

Hausinger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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confidence: 99%

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

Fellner

Desguin

Hausinger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: An Intriguing Cofactor In Lactate Racemasementioning

confidence: 88%

Lactate Racemization and Beyond

Desguin¹

2018

J Bacteriol Parasitol

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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.

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“…[44] A distinct mechanism for LarA, proton-coupled electron transfer (PCET), was proposed on the basis of quantum mechanics/molecular mechanics calculations. [46] This mechanism also proposes abstraction of the hydroxyl proton of lactate by a general base; however, it suggests this step occurs in concert with the homolytic cleavage of its C1À C2 bond and a 1-electron reduction of Ni(III) to Ni(II), yielding acetaldehyde and a carboxylate radical as intermediates (Scheme 8). Rotation about the carbonyl bond of acetaldehyde followed by Ni(II) oxidation and reconstitution of the C1À C2 bond permits either lactate isomer to form.…”

Section: Mechanism Of the Npn Cofactor In Lactate Racemizationmentioning

confidence: 99%

Biological Pincer Complexes

Nevarez

Turmo

et al. 2020

ChemCatChem

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At least two types of pincer complexes are known to exist in biology. A metal‐pyrroloquinolone quinone (PQQ) cofactor was first identified in bacterial methanol dehydrogenase, and later also found in selected short‐chain alcohol dehydrogenases of other microorganisms. The PQQ‐associated metal can be calcium, magnesium, or a rare earth element depending on the enzyme sequence. Synthesis of this organic ligand requires a series of accessory proteins acting on a small peptide, PqqA. Binding of metal to PQQ yields an ONO‐type pincer complex. More recently, a nickel‐pincer nucleotide (NPN) cofactor was discovered in lactate racemase, LarA. This cofactor derives from nicotinic acid adenine dinucleotide via action of a carboxylase/hydrolase, sulfur transferase, and nickel insertase, resulting in an SCS‐type pincer complex. The NPN cofactor likely occurs in selected other racemases and epimerases of bacteria, archaea, and a few eukaryotes.

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The Nickel‐Pincer Complex in Lactate Racemase Is an Electron Relay and Sink that acts through Proton‐Coupled Electron Transfer

Cited by 17 publications

References 38 publications

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

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

Lactate Racemization and Beyond

Biological Pincer Complexes

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