Uncovering a superfamily of nickel-dependent hydroxyacid racemases and epimerases

Abstract

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.

Characterization of a [4Fe-4S]-dependent LarE sulfur insertase that facilitates nickel-pincer nucleotide cofactor biosynthesis in Thermotoga maritima

“…The isomerization of D/L-lactate is catalyzed by lactate racemase (Lar), an enzyme that, in Lactobacillus plantarum , is a combination of the LarA protein and a tethered nickel-pincer nucleotide (NPN) cofactor ( 2 ). Homologs of LarA use NPN to catalyze racemization and epimerization reactions of a variety of α-hydroxy acid compounds other than lactate ( 3 ).…”

Metal Homeostasis in Pathogenic Streptococci

“…One exception, as mentioned above, is the urease and Ni transporter system that is encoded by S. salivarius [ 73 ]. Aside from this, there is only one known homolog of LarA (Ni-lactate racemase) found in S. pneumoniae [ 125 , 154 ] but, collectively, the role of Ni in the pathogenesis of Streptococci remains to be elucidated.…”

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

“…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. 11,12 The reaction mechanism of LarA Lp , the founding member of the LarA superfamily, has been extensively studied. 13 A protoncoupled hydride-transfer (PCHT) mechanism (Scheme 1b), where the left and right half-reactions of LarA Lp are analogous to the entire reactions of L-and D-lactate dehydrogenases, 1 has been proposed based on the enzyme structure, a substrate kinetic isotope effect, identification of the pyruvate intermediate, spectroscopic changes with an added substrate, and computational investigations.…”

“…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. , …”