6C1W

A tethered niacin-derived pincer complex with a nickel-carbon or sulfite-carbon bond in lactate racemase

Summary for 6C1W

• Entry DOI: 10.2210/pdb6c1w/pdb
• Descriptor: Lactate racemase, (4S)-5-methanethioyl-1-(5-O-phosphono-beta-D-ribofuranosyl)-4-sulfo-1,4-dihydropyridine-3-carbothioic S-acid, NICKEL (II) ION, ... (7 entities in total)
• Functional Keywords: lar, nickel transferase, lara, nickel, lactate, lactate racemization, lactate racemase, sulfite, sulfite-bond, pincer, isomerase, cofactor
• Biological source: Lactobacillus plantarum
• Total number of polymer chains: 3
• Total formula weight: 145172.18

Authors

Fellner, M.,Desguin, B.,Hausinger, R.P.,Hu, J. (deposition date: 2018-01-05, release date: 2018-03-14, Last modification date: 2024-11-06)

Primary citation

Rankin, J.A.,Mauban, R.C.,Fellner, M.,Desguin, B.,McCracken, J.,Hu, J.,Varganov, S.A.,Hausinger, R.P.
Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism.
Biochemistry, 57:3244-3251, 2018

PubMed Abstract: Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect ( k/ k of 3.11 ± 0.17) was established using 2-α-H-lactate, further supporting a PCHT mechanism. UV-visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.

PubMed: 29489337
DOI: 10.1021/acs.biochem.8b00100

Experimental method

X-RAY DIFFRACTION (2.398 Å)