Unveiling the mechanisms and biosynthesis of a novel nickel-pincer enzyme

Abstract

The nickel-pincer nucleotide (NPN) coenzyme, a substituted pyridinium mononucleotide that tri-coordinates nickel, was first identified covalently attached to a lysine residue in the LarA protein of lactate racemase. Starting from nicotinic acid adenine dinucleotide, LarB carboxylates C5 of the pyridinium ring and hydrolyzes the phosphoanhydride, LarE converts the C3 and C5 carboxylates to thiocarboxylates, and LarC incorporates nickel to form a C–Ni and two S–Ni bonds, during the biosynthesis of this cofactor. LarB uses a novel carboxylation mechanism involving the transient formation of a cysteinyl-pyridinium adduct. Depending on the source of the enzyme, LarEs either catalyze a sacrificial sulfur transfer from a cysteinyl side chain resulting in the formation of dehydroalanine or they utilize a [4Fe–4S] cluster bound by three cysteine residues to accept and transfer a non-core sulfide atom. LarC is a CTP-dependent enzyme that cytidinylylates its substrate, adds nickel, then hydrolyzes the product to release NPN and CMP. Homologs of the four lar genes are widely distributed in microorganisms, with some species containing multiple copies of larA whereas others lack this gene, consistent with the cofactor serving other functions. Several LarA-like proteins were shown to catalyze racemase or epimerase activities using 2-hydroxyacid substrates other than lactic acid. Thus, lactate racemase is the founding member of a large family of NPN-containing enzymes.

On the evolution of coenzyme biosynthesis

“…Apparently, it is recruited from NAD(P) + biosynthesis with the intermediate nicotinic acid adenine dinucleotide (NAAD) 54 serving as precursor. 55 An initial protein-bound cysteine-mediated activation of the pyridinium ring allows the introduction of the second carboxylate at C5 with CO 2 as C1 source. Carboxylation is accompanied by hydrolysis of the diphosphate moiety and cleavage of AMP.…”

Irreversible Inactivation of Lactate Racemase by Sodium Borohydride Reveals Reactivity of the Nickel–Pincer Nucleotide Cofactor

“…1,2 As the ninth nickel-dependent enzyme discovered in nature, LarA Lp utilizes a novel nickel−pincer nucleotide (NPN) cofactor (Scheme 1a) 3 to catalyze the isomerization reaction. 4,5 The coenzyme is synthesized from nicotinic acid adenine dinucleotide by consecutive actions of the LarB carboxylase/hydrolase, 6 LarE sulfur transferase, 7,8 and LarC nickel insertase. 9,10 The most prominent characteristic of the NPN cofactor is the direct bonding between Ni 2+ and the C4 atom in its pyridinium ring, representing the first C−Ni bond (other than transient intermediates) identified in a biological system.…”

“…LarA from Lactiplantibacillus plantarum (LarA Lp ) is a lactate racemase that interconverts the l - and d -enantiomers of the α-hydroxyacid. , As the ninth nickel-dependent enzyme discovered in nature, LarA Lp utilizes a novel nickel–pincer nucleotide (NPN) cofactor (Scheme a) to catalyze the isomerization reaction. , The coenzyme is synthesized from nicotinic acid adenine dinucleotide by consecutive actions of the LarB carboxylase/hydrolase, LarE sulfur transferase, , and LarC nickel insertase. , The most prominent characteristic of the NPN cofactor is the direct bonding between Ni 2+ and the C4 atom in its pyridinium ring, representing the first C–Ni bond (other than transient intermediates) identified in a biological system. Since the discovery of the NPN cofactor in 2015, the large LarA superfamily, previously denoted as the DUF2088 family, was shown to consist of highly diverse family members catalyzing racemization/epimerization reactions on a variety of α-hydroxyacids with many representatives catalyzing unknown reactions. , …”

Metallated dihydropyridinates: prospects in hydride transfer and (electro)catalysis

“…As the name lactate racemase suggests, LarA mediates the racemisation of lactate, and that process proceeds following hydride abstraction from lactate by the NPN cofactor, to permit a C–C bond rotation before HT from NPN furnishes the racemised lactate molecule. 29 There are also cofactors that facilitate HT in biology via O–H and N–H bond chemistries; they are outside the scope of this Perspective and so those cofactors will not be discussed. 25,30–33…”