Nickel-pincer cofactor biosynthesis involves LarB-catalyzed pyridinium carboxylation and LarE-dependent sacrificial sulfur insertion

Abstract

The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.

Keywords: lactate; nicotinic acid dinucleotide; racemase; sacrificial enzyme.

Fig. 1.

Identification of the substrate of LarB. (A) Structure of the (SCS)Ni(II) pincer cofactor covalently bound to L. plantarum LarA and synthesized by LarB, LarC, and LarE. (B) Lar activity generated upon addition of LarA apoprotein to solutions incubated for 2 h with LarE (5 µM), LarC (5 µM), MgCl₂ (20 mM), ATP (5 mM), and CoA (1 mM) in Tris⋅HCl buffer (100 mM; pH 8) along with (where indicated) LarB (1 µM), NaMN (0.5 mM), NaAD (0.5 mM), NaHCO₃ (10 mM), and NiCl₂ (0.1 mM). (C) LarB reaction was performed for 10 min in Tris⋅HCl buffer (100 mM; pH 8) with MgCl₂ (4 mM) along with (where indicated) LarB (0.5 µM), NaMN (0.2 mM), NaAD (0.2 mM), NaHCO₃ (100 mM), ATP (5 mM), and NiCl₂ (0.1 mM) before inactivation and incubation for 1 h with LarE, LarC, MgCl₂, ATP, and CoA and addition of LarA apoprotein as in B. Error bars represent the SD (n = 4).

Fig. 2.

Identification of the LarB and LarE products. (A) LC-ESI-MS chromatographs in positive-ionization mode of NaAD mixed with MgCl₂ (5 mM) and NaHCO₃ (10 mM) in the absence and presence of LarB. (B) Integration of the 0.5- to 0.6-min region of A with NaHCO₃ (10 mM) or NaH¹³CO₃ (10 mM). (C) Integration of the 0.5- to 0.7-min region of a separate LC-ESI-MS chromatograph in positive-ionization mode after the LarE reaction with P2CMN or P2CMN*. The identities of the compounds as determined by LC-ESI-MS/MS (SI Appendix, Figs. S3, S6, and S7) are indicated next to the corresponding peaks, where the asterisk (*) indicates the presence of ¹³C.

Fig. 3.

Sulfur transfer from LarE to P2CMN. (A) Percentage of each form of LarE as observed by LC-MS-ESI before (Control) and 0, 15, 30, and 45 min after addition of the LarB product (SI Appendix, Fig. S8). Error bars represent the SD (n = 2). (B) LC-ESI-MS of a doubly charged tryptic peptide of LarE before (Upper) and 30 min after (Lower) addition of P2CMN. The sequences of the peptides analyzed by LC-ESI-MS/MS (SI Appendix, Fig. S10) are indicated above the corresponding peaks.

Fig. 4.

LarC requirement and Lar activity generation. (A) Lar activity generated upon addition of LarA apoprotein to solutions incubated for 2 h with NaAD (0.5 mM), NaHCO₃ (10 mM), LarB (1 µM), LarE (5 µM), MgCl₂ (20 mM), ATP (5 mM), and CoA (1 mM) in Tris⋅HCl buffer (100 mM; pH 8) along with (where indicated) LarC and NiCl₂ (0.1 mM). (B) Time course of the in vitro activation of L. plantarum LarA in the absence (−Sulfite) or presence (+Sulfite) of 10 µM sulfite. Error bars represent the SD (n = 3).

Fig. 5.

Overall pathway for synthesis of the LarA cofactor.