Identification and molecular characterization of a metagenome-derived L-lysine decarboxylase gene from subtropical soil microorganisms

Abstract

L-lysine decarboxylase (LDC, EC 4.1.1.18) is a key enzyme in the decarboxylation of L-lysine to 1,5-pentanediamine and efficiently contributes significance to biosynthetic capability. Metagenomic technology is a shortcut approach used to obtain new genes from uncultured microorganisms. In this study, a subtropical soil metagenomic library was constructed, and a putative LDC gene named ldc1E was isolated by function-based screening strategy through the indication of pH change by L-lysine decarboxylation. Amino acid sequence comparison and homology modeling indicated the close relation between Ldc1E and other putative LDCs. Multiple sequence alignment analysis revealed that Ldc1E contained a highly conserved motif Ser-X-His-Lys (Pxl), and molecular docking results showed that this motif was located in the active site and could combine with the cofactor pyridoxal 5'-phosphate. The ldc1E gene was subcloned into the pET-30a(+) vector and highly expressed in Escherichia coli BL21 (DE3) pLysS. The recombinant protein was purified to homogeneity. The maximum activity of Ldc1E occurred at pH 6.5 and 40°C using L-lysine monohydrochloride as the substrate. Recombinant Ldc1E had apparent Km, kcat, and kcat/Km values of 1.08±0.16 mM, 5.09±0.63 s-1, and 4.73×103 s-1 M-1, respectively. The specific activity of Ldc1E was 1.53±0.06 U mg-1 protein. Identifying a metagenome-derived LDC gene provided a rational reference for further gene modifications in industrial applications.

Fig 1. Multiple sequence alignment of the Ldc1E protein and other LDCs.

Colored boxes indicate the secondary structures, which were predicted by PSIPRED Server, with helix and sheets represented by red and yellow boxes, respectively. Dashes indicate gaps. Conserved LDC decarboxylase motifs are indicated in the blue line. Red and blue asterisks show the conserved residues in PLP binding sites and the predicted residues in L-lysine binding sites, respectively. The sequences from top to bottom are E. coli K-12 LDC (P52095.2), E. coli O157:H7 LDC (P0A9H4.1), S. enterica subsp. LT2 LDC (P0A1Z0.1), H. alvei LDC (P05033.1) and A. hydrophila AL06-06 (WP_017408594.1).

Fig 2. Phylogenetic tree analysis of the Ldc1E protein with other known LDCs based on the amino acid sequences.

A phylogenetic tree was constructed using the neighbor-joining method with MEGA 6.0, and 1,000 bootstrap replicates were indicated at branching points. Ldc1E was shown with a solid triangle. The tree also shows the GenBank accession number and original genus of other LDCs, with the scale bar representing the number of changes per amino acid position.

Fig 3. Homology modeling structure of LDC and Ldc1E.

(A) Superposition of the Ldc1E monomer (yellow) on the LDC from E. coli K-12 (PDB ID: 3n75). (B) Ribbon representation of Ldc1E homo-decamer. (C) Ball-and-stick representation of the docking models of Ldc1E with PLP. Lys367 was a catalytic residue located in the active center, and the Trp333, Ser364, and His366 stabilized the PLP structure. (D) Ball-and-stick representation of the docking models of Ldc1E with L-lysine, where Lys367, Val524, and Glu526 were probably substrate binding sites.

Fig 4. 12% (w/v) SDS-PAGE analysis of recombinant Ldc1E.

Lane 1, molecular mass standards. Lane 2, total protein of E. coli BL21 (DE3) pLysS harboring empty pET30a(+) (control). Lane 3, total protein of E. coli BL21 (DE3) pLysS harboring the recombinant ldc1E in pET30a(+). and Lane 4, protein was purified using the Ni-NTA column method. The black arrow indicates the recombinant Ldc1E.

Fig 5. RP-HPLC profile of the reaction product from the decarboxylation of L-lysine HCl substrate under Ldc1E catalysis.

The enzymatic product was derivatized with dansyl chloride.

Fig 6. Effects of temperature and pH on the activity and stability of Ldc1E.

(A) Optimum reaction temperature of the recombinant Ldc1E. The enzyme activity was measured at various temperatures from 20°C to 60°C with 5°C intervals in 0.2 M Na₂HPO₄/0.1 M citric acid buffer (pH 6.5). Relative activity of 100% represents the specific activity of 1.50±0.06 U mg⁻¹ protein. (B) Effect of temperature on the enzymatic activity of recombinant Ldc1E. Relative activity of 100% represents the specific activity of 1.53±0.06 U mg⁻¹ protein. (C) Effect of pH on the enzymatic activity of recombinant Ldc1E. The enzyme activity was measured in 0.2 M Na₂HPO₄/0.1 M citric acid (4.0–8.0) and 0.1 M glycine–NaOH (8.0–10.0) at 40°C. Relative activity of 100% represents the specific activity of 1.53±0.06 U mg⁻¹ protein. (D) Effect of stable pH on the enzymatic activity of the recombinant Ldc1E. Relative activity of 100% represents the specific activity of 0.93±0.05 U mg⁻¹ protein.

Fig 7. Substrate specificity test of L-lysine-HCl, L-arginine-HCl, and L-ornithine-HCl.

One unit was defined as the enzyme consumption of 10 μg L-lysine-HCl/L-arginine-HCl/L-ornithine-HCl per minute, respectively.