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Abstract LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.
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Biological Pincer Complexes
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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- Award ID(s):
- 1807073
- PAR ID:
- 10233803
- Date Published:
- Journal Name:
- ChemCatChem
- Volume:
- 12
- ISSN:
- 1867-3880
- Page Range / eLocation ID:
- 4242-4254
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)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.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/doi.org/10.1002/cctc.202000575
