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Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

ABSTRACT: The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarA_Lp) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarA_Lp, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH₄) irreversibly inactivates LarA_Lp accompanied by a rapid color change of the enzyme. The altered ultraviolet-visible spectra during NaBH₄ titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the irreversible inactivation. High resolution structures of LarA_Lp revealed a substantially weakened C-Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarA_Lp inactivation by NaBH₄ that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.

SUBMITTER: Gatreddi S

PROVIDER: S-EPMC10599654 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

Gatreddi Santhosh S Sui Dexin D Hausinger Robert P RP Hu Jian J

ACS catalysis 20230110 2

The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarALp) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarALp, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydr ...[more]

PMID: 37886035

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Project description:The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the Lactiplantibacillus plantarum lar genes are cloned into the Duet expression system and cultured in Escherichia coli, they confer lactate racemase activity to the cells. By replacing L. plantarum larA with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in E. coli, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins.ImportanceEnzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in Escherichia coli and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism.

Project description:The 15e square-planar complexes [Co(PCPMe-iPr)Cl] (2a) and [Co(PCP-tBu)Cl] (2b), respectively, react readily with NaBH4 to afford complexes [Co(PCPMe-iPr)(η2-BH4)] (4a) and [Co(PCP-tBu)(η2-BH4)] (4b) in high yields, as confirmed by IR spectroscopy, X-ray crystallography, and elemental analysis. The borohydride ligand is symmetrically bound to the cobalt center in η2-fashion. These compounds are paramagnetic with effective magnetic moments of 2.0(1) and 2.1(1) μB consistent with a d7 low-spin system corresponding to one unpaired electron. None of these complexes reacted with CO2 to give formate complexes. For structural and reactivity comparisons, we prepared the analogous Ni(II) borohydride complex [Ni(PCPMe-iPr)(η2-BH4)] (5) via two different synthetic routes. One utilizes [Ni(PCPMe-iPr)Cl] (3) and NaBH4, the second one makes use of the hydride complex [Ni(PCPMe-iPr)H] (6) and BH3·THF. In both cases, 5 is obtained in high yields. In contrast to 4a and 4b, the borohydride ligand is asymmetrically bound to the nickel center but still in an η2-mode. [Ni(PCPMe-iPr)(η2-BH4)] (5) loses readily BH3 at elevated temperatures in the presence of NEt3 to form 6. Complexes 5 and 6 are both diamagnetic and were characterized by a combination of 1H, 13C{1H}, and 31P{1H} NMR, IR spectroscopy, and elemental analysis. Additionally, the structure of these compounds was established by X-ray crystallography. Complexes 5 and 6 react with CO2 to give the formate complex [Ni(PCPMe-iPr)(OC(C=O)H] (7). The extrusion of BH3 from [Co(PCPMe-iPr)(η2-BH4)] (4a) and [Ni(PCPMe-iPr)(η2-BH4)] (5) with the aid of NH3 to yield the respective hydride complexes [Co(PCPMe-iPr)H] and [Ni(PCPMe-iPr)H] (6) and BH3NH3 was investigated by DFT calculations showing that formation of the Ni hydride is thermodynamically favorable, whereas the formation of the Co(II) hydride, in agreement with the experiment, is unfavorable. The electronic structures and the bonding of the borohydride ligand in [Co(PCPMe-iPr)(η2-BH4)] (4a) and [Ni(PCPMe-iPr)(η2-BH4)] (5) were established by DFT computations.

Dataset Information

Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

Publications

Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

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