# Nickel-pincer nucleotide enzymes

> **NIH NIH R01** · MICHIGAN STATE UNIVERSITY · 2021 · $405,329

## Abstract

Project Summary/Abstract
The nickel-pincer nucleotide (NPN) cofactor is a newly identified coenzyme discovered in lactate racemase
(LarA) from Lactobacillus plantarum. Synthesis of the active enzyme requires the participation of three accessory
proteins that act in sequence: LarB carboxylates the pyridinium ring and hydrolyzes the phosphoanhydride of
nicotinic acid adenine dinucleotide, LarE converts the two pyridinium ring carboxylates to thiocarboxylates, and
LarC inserts nickel (forming two S-Ni and one C-Ni bonds) during synthesis of the novel cofactor. Genes
encoding these four proteins are widely distributed in microorganisms associated with the human microbiome
and among human pathogens. The long-term objective of the effort described here is to advance significantly
our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor.
Two specific aims will achieve this objective: (1) characterize the components of the NPN biosynthetic systems
and (2) identify the roles of the NPN cofactor in lactate racemase and additional enzymes. Investigations of LarB
will define the structure and mechanism of this pyridinium ring carboxylase/phosphoanhydride hydrolase. Studies
of a multi-cysteine and probable [4Fe4S]-containing form of LarE will establish whether it operates by a catalytic
sulfur-transfer mechanism, in contrast to the sacrificial LarE of L. plantarum with its single active site cysteine
that converts to dehydroalanine. Structural and mechanistic analysis of the CTP-dependent nickel-inserting LarC
will elucidate how this protein installs nickel into the cofactor. The geometry of lactate binding to L. plantarum
LarA will be defined, the full range of substrates used by this enzyme will be established, and substrates will be
identified for alternative LarA-like proteins. Proteins that covalently bind the NPN cofactor will be identified and
characterized using innovative chemistry that reacts the coenzyme with a fluorescent tag. Radioactive nickel
(63Ni) and 14C-nicotinic acid also will be used to label new NPN cofactor-binding proteins, followed by mass
spectrometry, bioinformatics, and biochemical studies to identify the functions of these novel enzymes. The
findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel-
pincer cofactors in bacteria, including those important to human health, with implications for identification of
potential antimicrobial drug targets.

## Key facts

- **NIH application ID:** 10141260
- **Project number:** 5R01GM128959-03
- **Recipient organization:** MICHIGAN STATE UNIVERSITY
- **Principal Investigator:** ROBERT P HAUSINGER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $405,329
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141260

## Citation

> US National Institutes of Health, RePORTER application 10141260, Nickel-pincer nucleotide enzymes (5R01GM128959-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10141260. Licensed CC0.

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