8EZI

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

(2023) ACS Catal 13: 1441-1448

• PubMed: 37886035 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1021/acscatal.2c05461
• Primary Citation of Related Structures:  
  8EZF, 8EZH, 8EZI


• PubMed 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 (NaBH ₄ ) irreversibly inactivates LarA _Lp accompanied by a rapid color change of the enzyme. The altered ultraviolet-visible spectra during NaBH ₄ 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 NaBH ₄ that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.

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Organizational Affiliation: 

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, United States.