GyrI-like proteins catalyze cyclopropanoid hydrolysis to confer cellular protection

Abstract

GyrI-like proteins are widely distributed in prokaryotes and eukaryotes, and recognized as small-molecule binding proteins. Here, we identify a subfamily of these proteins as cyclopropanoid cyclopropyl hydrolases (CCHs) that can catalyze the hydrolysis of the potent DNA-alkylating agents yatakemycin (YTM) and CC-1065. Co-crystallography and molecular dynamics simulation analyses reveal that these CCHs share a conserved aromatic cage for the hydrolytic activity. Subsequent cytotoxic assays confirm that CCHs are able to protect cells against YTM. Therefore, our findings suggest that the evolutionarily conserved GyrI-like proteins confer cellular protection against diverse xenobiotics via not only binding, but also catalysis.

Fig. 1

Structures of cyclopropapyrroloindole compounds and characterization of cyclopropanoid cyclopropyl hydrolases (CCHs). a Structures of yatakemycin (YTM), CC-1065, and duocarmycins. 1 L, 1 M, and 1 R indicate the three subunits of YTM, respectively. The IC₅₀ reveals the cytotoxicity against L1210 cell line. b Genetic investigation of ytkR7 by HPLC analysis of the fermentation products (UV at 383 nm). (i) wild-type Streptomyces sp. TP-A0356; (ii) the ΔytkR7 mutant Streptomyces sp. TG1310; (iii) the ΔytkR7 mutant complemented with the ytkR7 gene in trans; and (iv) the ΔytkR7 mutant complemented with the c10R6 gene from S. zelensis NRRL 11183. c Structures of compounds 5, 6, and 7. d Biochemical characterization of YtkR7 with YTM as substrate. (i) YTM dissolved in the reaction buffer; (ii) boiled YtkR7; (iii) YtkR7; and (iv) standard of 5. e Biochemical assays of the other selected CCHs using YTM as substrate. (i) C10R6; (ii) lin2189 from Listeria innocua Clip11262; (iii) ETI84332.1 from Streptococcus anginosus DORA_7 (from the human microbiota); and (iv) MA1133 from Methanosarcina acetivorans C2A. f Characterization of substrate specificity of the CCH protein C10R6 by HPLC analysis (UV at 374 nm). (i) CC-1065 dissolved in the reaction buffer; (ii) C10R6 with CC-1065 as substrate; (iii) the fermentation products of S. zelensis NRRL 11183; and (iv) standard of 7

Fig. 2

Structural and molecular dynamics (MD) simulation analyses of lin2189-YTM. a The molecular details of the lin2189 E157A/E185L-YTM crystal structure. Green ball-and-stick cartoon, the substrate (YTM); cyan spheres, polar residues; yellow spheres, aromatic residues; and blue spheres, hydrophobic residues. b Interactions between native lin2189 and YTM from MD simulations. Black dash, H-bond interactions. c The normalized interaction frequency of native lin2189-YTM calculated from unbiased MD simulations. d The intrinsic mobility of lin2189 calculated from the principal component analysis (PCA). The cyan vector length correlates with the domain-motion scale. Yellow sphere, the substrate molecule (YTM) from docking; red region, the catalytic pocket. PCA analysis showed that loops A, B and C next to the catalytic pocket are very flexible. e The substrate entrance pathway sampled by metaMD simulations. Yellow stick, final pose of the substrate YTM. Green stick, the catalytic trait in the binding pocket. Blue spheres, the mass center of the substrate molecule along the simulation trajectory. f The product leaving pathway sampled by metaMD simulations. g The catalytic residues and aromatic cage of CCHs are highly conserved. YtkR7, and the 1695 similar proteins were used to create the sequence logo. The residue numbers correspond to those of lin2189

Fig. 3

The catalytic mechanism of CCHs. a In an apo CCH, the three loops (loop A, B, and C) next to the catalytic pocket are very flexible and the size of the pocket can be fluctuated to a large extent. E157 is protonated, whereas E185 is deprotonated. b The entry of substrate into the CCH leads to the outward shift of loop C, resulting in a large space in the binding pocket. This facilitates the substrate molecule entering the catalytic region. c After substrate situating at the catalytic site and in an activated binding mode. E185 acts as a nucleophile, attacking the methylene group in the cyclopropyl ring. This leads to transformation of the quinone moiety into a deprotonated phenol. d The deprotonated phenol moiety of the transitional substrate acquires a proton from a water molecule. e With the assistance of E157, the transitional state is hydrolyzed by a water molecule. f Finally, with the outward fluctuations of loop B and loop C, the product leaves catalytic pocket. The E185 residue returns to its deprotonated state

Fig. 4

CCHs can protect E. coli against the potent DNA-alkylating agent YTM. Four selected CCH proteins include YtkR7, lin2189, ETI84332.1, and MA1133, which cover from both bacteria and archaea (from diverse environments, including soil and the human microbiota)