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

Abstract

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...

Biosynthesis of the nickel-pincer nucleotide cofactor of lactate racemase requires a CTP-dependent cyclometallase

“…Lactate racemase (LarA), the ninth discovered nickeldependent enzyme (1), was shown to contain a newly identified cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN), 5 covalently attached to an active-site lysine residue (2). Synthesis of NPN occurs through a pathway involving three proteins encoded in the lar operon, i.e.…”

“…NPN biosynthesis begins with LarB, a carboxylase/hydrolase that produces pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN) from nicotinic acid adenine dinucleotide (NaAD) (4). P2CMN is converted into pyridinium-3,5-bisthiocarboxylic acid (P2TMN) by LarE through two successive sacrificial sulfur transfer reactions (5). Finally, LarC inserts nickel and forms the final NPN cofactor with its five-membered nickellacycle structure (Fig.…”

“…4 Co-senior authors. 5 The abbreviations used are: NPN, nickel-pincer nucleotide; P2CMN, pyridinium-3,5-biscarboxylic acid mononucleotide; P2TMN, into pyridinium-3,5bisthiocarboxylic acid; NaAD, nicotinic acid adenine dinucleotide; PRF, programmed ribosomal frameshift; BME, ␤-mercaptoethanol; FeGP, ironguanylylpyridinol; PDB, Protein Data Bank; r.m.s.d., root mean square deviation; PAR, 4-(2-pyridylazo)resorcinol.…”

Structure, function, and biosynthesis of nickel‐dependent enzymes

“…The enzyme catalyzes the AMPylation of the pyridinium carboxyl groups followed by a sulfur transfer, via the conserved cysteine 176 present in the C‐terminal part of the protein as a sulfur donor to convert P2CMN to P2TMN. This hypothesis has been recently proven thanks to the determination of the crystal structures of LarE from L. plantarum and its C176A mutant, in their apo‐form and in complex with coenzyme A (CoA) . This in vitro study revealed the essential role of Cys176 as the catalytic site for the sulfur insertion, as well as the role of CoA persulfilde in the regeneration of active LarE, although the physiological relevance of this cycling mechanism remains unclear.…”

“…Among the lar gene cluster, LarB is responsible for the biosynthesis of pyridinium 3,5‐biscarboxylic acid mononucleotide (P2CMN) by catalyzing the carboxylation of nicotinic acid adenine dinucleotide (NaAD) . Subsequently, LarE converts P2CMN into pyridinium 3,5‐biscarboxylic acid mononucleotide (P2TMN) via two sulfur transfer reactions and LarC provides the nickel to generate the active form of the pincer cofactor that will bind to LarA . LarE belongs to the PP‐loop pyrophosphatase family and possesses two independent domains: a N‐terminal domain containing the conserved PP‐loop SGGXDS motif and a C‐terminal domain which is not common to any member of this N‐type ATPase family, revealing a double function for LarE.…”