Crystal structure determination of the halogenase CtcP from Streptomyces aureofaciens

Abstract

Chlortetracycline (CTC), a derivative of tetracycline (TC), is a broadly used antibiotic that inhibits the synthesis of bacterial proteins by competing with the A-site tRNA on ribosomes. A recent study showed that during the biosynthesis of CTC in Streptomyces aureofaciens, the halogenase CtcP catalyzes the final chlorination reaction and transforms TC into CTC. However, the structure of this fundamental enzyme is still lacking. Here, selenomethionine-derivatized CtcP from S. aureofaciens was overexpressed and purified and its structure was determined at 2.7 Å resolution. The structure of CtcP reveals the conserved monooxygenase domain shared by all flavin-dependent halogenases and a unique C-terminal domain. Although FAD was not observed in the structure, the monooxygenase domain has a conserved FAD-binding pocket and active center. The C-terminal domain displays an α-helical bundle fold, which could contribute to substrate specificity. This work provides a molecular basis for enzyme engineering to improve the industrial production of CTC.

Keywords: CtcP; Streptomyces aureofaciens; antibiotics; chlortetracycline; crystal structure; halogenases.

Figure 1

The crystal structure of CtcP. The crystal structure of CtcP from S. aureofaciens is shown in ribbon representation in two different views. The N-terminal (green) and C-terminal (blue) domains of CtcP are color-coded. α-Helix 10 from the N-terminal domain and α-helices 11–17 from the C-terminal domain are labeled. Due to the slight difference among the four molecules in the asymmetric unit, only residues 15–551 were built in this chosen copy.

Figure 2

Structural comparison of CtcP with PltM. Two different views of a structural comparison between the P. protegens halogenase PltM in complex with phloroglucinol (PDB entry 6bza) and CtcP (this study). The CtcP protein is rainbow-colored, while PltM is colored gray.

Figure 3

Structural comparison of the FAD- and substrate-binding sites. (a) All of the essential residues which coordinate FAD in both CtcP and PltM are shown in stick representation. CtcP is colored green, while PltM is colored gray. The FAD molecule and the Cl⁻ ion are from the model of PltM. (b) Comparison of the substrate-binding site in CtcP and PltM. The enzymatic active-center residues (Lys87 in PltM and Lys98 in CtcP) and the phloroglucinol from the PltM structure are shown in stick representation. CtcP is colored green and blue, while PltM is colored gray.