Crystal structure of CmnB involved in the biosynthesis of the nonproteinogenic amino acid L-2,3-diaminopropionic acid

Abstract

L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products, including capreomycin, viomycin, zwittermicin, staphyloferrin and dapdiamide. A previous study reported that CmnB and CmnK are two enzymes that are involved in the formation of L-Dap in the biosynthesis of capreomycin. CmnB catalyzes the condensation reaction of O-phospho-L-serine and L-glutamic acid to generate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid, which subsequently undergoes oxidative hydrolysis via CmnK to generate the product L-Dap. Here, the crystal structure of CmnB in complex with the reaction intermediate PLP-α-aminoacrylate is reported at 2.2 Å resolution. Notably, CmnB is the second known example of a PLP-dependent enzyme that forms a monomeric structure in crystal packing. The crystal structure of CmnB also provides insights into the catalytic mechanism of the enzyme and supports the biosynthetic pathway of L-Dap reported in previous studies.

Keywords: CmnB; PLP-dependent enzymes; capreomycin biosynthesis; l-2,3-diaminopropionic acid; l-Dap.

Figure 1

Schematic representation of the formation of the l-Dap moieties in capreomycin biosynthesis.

Figure 2

Structure of CmnB. (a) Ribbon diagram of the predicted structure of CmnB generated by AlphaFold. Regions with confidence scores (pLDDT) between 0 and 100 are colored red (<50), yellow (60), green (70), cyan (80) and blue (>90). (b) Ribbon diagram of the crystal structure of CmnB. The small and large domains are colored blue and gray, respectively. PLP-α-aminoacrylate between the two domains is depicted in stick representation in yellow. The composite (2mF _o − F _c) omit map contoured at 1.0σ is colored green.

Figure 3

Structure-based sequence alignment of CmnB and SbnA. Aligned residues are colored on the basis of the level of conservation (a red background shows strict identity, red letters show similarity and a blue frame shows similarity across groups). The corresponding secondary structures are depicted above the sequences. The residues involved in binding PLP, α-aminoacrylate and l-Glu are labeled under the sequence alignment by circles, diamonds and triangles, respectively.

Figure 4

Structural superposition of CmnB (blue) with SbnA (PDB entry 5d85; pink). (a) The overall structures of CmnB and SbnA. (b) Local view of the PLP-α-aminoacrylate-binding cavity. (c) The putative l-Glu-binding pocket. Protein–ligand interactions are depicted as yellow dotted lines.

Figure 5

Structural comparison of CmnB with SbnA. (a) Superposition of the overall structures of the CmnB monomer (blue) and the SbnA dimer (pink and purple). Two regions involved in SbnA dimerization are highlighted with a red background. (b, c) Local views of the dimerization regions. The protein–protein interactions in (b) are depicted as yellow dotted lines.