Lincosamide antibiotics, including lincomycin A and celesticetin, are natural antibacterial compounds effective against Gram-positive bacteria¹. The structures of lincosamides feature an unusual thiooctose connected with N-methyl-trans-4-propyl-L-proline (N-methyl-PPL) or N-methyl-L-proline (N-methyl-Pro) via an amide bond². Previous analyses of the structure-activity relationships of lincosamides indicated that the condensation between thiooctose and N-methyl-PPL or N-methyl-Pro is essential for their antibacterial activity³. Most of the steps in the biosynthetic pathways of lincomycin A and celesticetin have been identified⁴. In the biosynthesis of these compounds, the novel condensation enzymes LmbD and CcbD catalyze the condensation reaction between a carrier protein-tethered PPL or Pro and ergothioneine (EGT) S-conjugated thiooctose to form the pharmacophore of lincosamides⁵. Although the characterization of CcbD clearly indicated that the enzyme catalyzes the amide bond formation reaction, its biochemical properties, structure, and detailed reaction mechanism remained unclear. Particularly, the catalytic residues of the enzymatic reaction and the means by which the enzyme recognizes the carrier protein were still unknown.

To clarify the substrate selectivity of the enzyme, we first performed enzymatic reactions using CcbD with various acyl-carrier proteins, including different chain lengths of fatty-acyl carrier proteins and various amino acyl-carrier proteins as substrates. The results revealed that CcbD strongly recognizes the carrier protein, and shows substrate tolerance toward the acyl moiety structures. We also found that the products of these enzymatic reactions have novel structures that have not been previously identified in nature.

To elucidate the structure-function details of CcbD, we performed an X-ray crystallographic analysis. CcbD has a novel overall structure consisting of three domains, including the α/β-fold domain, four anti-parallel β-sheets, and the C-terminal five α-helices domain, and is quite different from the structures of previously analyzed enzymes. Interestingly, despite the lack of sequence similarity, the α/β-fold domain of CcbD shares weak structural similarity to those of cysteine proteases and phytochelatin synthases.

We also obtained the complex structure of the enzyme and substrate. The structure and following mutagenesis experiments indicated the catalytic residues and the mechanism of substrate recognition. In the active site, Cys17, His131, and Glu148 play catalytic roles to form the PPL/Pro-tethered Cys17 intermediate.

To determine how CcbD recognizes the carrier protein in detail, we solved the complex structure. CcbD and the carrier protein were covalently cross-linked by using 1,2-bis(maleimido)ethane (BMOE). The complex structure revealed that CcbD recognizes the carrier protein through salt bridges, hydrogen bond interactions, and hydrophobic interactions. A mutagenesis study of the interface residues between CcbD and the carrier protein indicated that the salt bridges between Arg312, Arg316, and Arg324 in CcbD and Glu47, Glu40, and Glu41 in the carrier protein, respectively, are important for the interaction.

In summary, we performed structural and functional analyses of CcbD-catalyzed amide bond formation, which is required for the biological activity of lincosamide antibiotics. The reaction catalyzed by CcbD is mechanistically and structurally distinct from the previously identified carrier protein-dependent amide bond formation reactions. Our analyses of CcbD have provided important new insights into the diversity of amide bond formation reactions in nature. Furthermore, we have successfully generated a series of non-natural, novel lincosamide compounds by using substrate analogues. Further engineering of the substrate specificity and the enzyme reactivity of CcbD is expected to generate biocatalysts that can be used for the generation of unnatural lincosamides with various acyl and sugar moieties for future drug discovery.

More details of this work can be found here: “Molecular basis for carrier protein-dependent amide bond formation in the biosynthesis of lincosamide antibiotics."  in Nature Catalysis, https://doi.org/10.1038/s41929-023-00971-y.

References

1. Janata, J., Kamenik, Z., Gazak, R., Kadlcik, S. & Najmanova, L. Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products. Nat. Prod. Rep. 35, 257-289 (2018).

2. Zhang, D., Tang, Z. & Liu, W. Biosynthesis of lincosamide antibiotics: reactions associated with degradation and detoxification pathways play a constructive role. Acc. Chem. Res. 51, 1496-1506 (2018).

3. Janata, J. et al. Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism. PLoS One 10, e0118850 (2015).

4. Wang, S.A. et al. Studies of lincosamide formation complete the biosynthetic pathway for lincomycin A. Proc. Natl. Acad. Sci. U S A 117, 24794-24801 (2020).

5. Zhao, Q., Wang, M., Xu, D., Zhang, Q. & Liu, W. Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. Nature 518, 115-119 (2015).