Structural insights into the catalytic mechanism of a sacrificial sulfur insertase of the N-type ATP pyrophosphatase family, LarE

Abstract

The lar operon in Lactobacillus plantarum encodes five Lar proteins (LarA/B/C/D/E) that collaboratively synthesize and incorporate a niacin-derived Ni-containing cofactor into LarA, an Ni-dependent lactate racemase. Previous studies have established that two molecules of LarE catalyze successive thiolation reactions by donating the sulfur atom of their exclusive cysteine residues to the substrate. However, the catalytic mechanism of this very unusual sulfur-sacrificing reaction remains elusive. In this work, we present the crystal structures of LarE in ligand-free and several ligand-bound forms, demonstrating that LarE is a member of the N-type ATP pyrophosphatase (PPase) family with a conserved N-terminal ATP PPase domain and a unique C-terminal domain harboring the putative catalytic site. Structural analysis, combined with structure-guided mutagenesis, leads us to propose a catalytic mechanism that establishes LarE as a paradigm for sulfur transfer through sacrificing its catalytic cysteine residue.

Keywords: ATP pyrophosphatase; Lar protein; catalysis; crystal structure; thiolation.

Fig. 1.

Ni-pincer LarA-cofactor biosynthesis highlighting the sacrificial sulfur transfer reactions of LarE. Dha, dehydroalanine; PCTMN, pyridinium-3-carboxy-5-thiocarboxylic acid mononucleotide.

Fig. 2.

Quaternary structure and metal binding of LarE. (A) Side view of the quaternary hexameric structure of LarE. Individual chains are colored as A = cyan, B = magenta, C = yellow, D = orange, E = blue, and F = green. A metal (gray sphere) binding site (along with a water molecule represented as a red sphere) is depicted for each trimer. (B) Top view of the hexamer.

Fig. 3.

Sequence alignment of LarE homologs. Numbering is based on LarE_Lp sequence. Residues are depicted white on black for invariant residues and black on gray for conserved ones. Ef, Enterococcus faecalis; Lp, L. plantarum; Pp, Pediococcus pentosaceus; Se, Staphylococcus epidermidis.

Fig. 4.

Domain structure and phosphate binding in LarE. (A) Domain structure of LarE. The head domain is shown in cyan, a flexible linker with Cys176 is shown in dark green, and the ATP PPase domain is shown in light blue with the conserved ²⁶SGGxDS³¹ PP-loop motif illustrated. Helices are labeled α1–α14, and sheets are labeled β1–β7. The disordered fragment (residues 126–147) is depicted as a dashed line. (B) ATP PPase domain of LarE in light blue is aligned with the ATP PPase domain of E. coli GMP synthetase (PDB ID code 1GPM) (9) in pink. The PP-loop residues are colored darker. (C) Close-up view of a phosphate molecule bound to the LarE head domain. H-bonds are illustrated as red dashed lines.

Fig. 5.

Comparison of ATP-bound, AMP-bound, and substrate-free LarE structures. ATP-bound structure of LarE (A; PDB ID code 5UDS) compared with AMP-bound structure of LarE (B; PDB ID code 5UDT). The 2F_O-F_C maps are shown as blue meshes at 1σ. Interacting residues are shown in stick mode. Carbon atoms of ATP and AMP are depicted in purple and teal, respectively. An Mg atom is shown as a green sphere, with its chelation represented as black dashed lines. Hydrogen bonds are indicated as red dashed lines. (C) Comparison of residues 122–148 loop in the absence (alternative substrate-free LarE PDB ID code 5UNM ribbon in yellow) and presence of AMP.

Fig. 6.

Substrate analog NMN binding to the C-terminal head domain of LarE. (A) NMN-bound structure of LarE (PDB ID code 5UDR). The F_O-F_C map is shown as green mesh at 2.5 σ for the model before adding the ligand. Carbon atoms of NMN are depicted in magenta. Hydrogen bonds are illustrated as red dashed lines. (B) Activity measurement of LarE variants compared with WT and with the addition of just buffer without LarE protein. The relative activity was the mean of three to nine repeats from up to three independent purifications, and the error bar represents the SD.

Fig. 7.

Structural model of P2CMN-bound LarE. (A) Carbon atoms of P2CMN and AMP are illustrated in gray and teal, respectively; the black dashed lines represent distances between the P2CMN, AMP, and sulfur atom of Cys176, as well as the distances from the C_α of Cys176 to Glu61 and Arg181. Red dashed lines indicate hydrogen bonds between Arg181 and Glu200. (B) Activity measurement of LarE variants compared with WT and with the addition of just buffer without LarE protein. The relative activity was the mean of four repeats, and the error bar represents the SD.

Fig. 8.

Proposed LarE mechanistic reaction scheme. PCTMN, pyridinium-3-carboxy-5-thiocarboxylic acid mononucleotide.