A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Abstract

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Keywords: iron–sulfur cluster; leucyl transfer reaction; natural product; tRNA-dependent enzymes.

Fig. 1.

The proposed biosynthetic pathway of BS. CMP: Cytidine 5-monophosphate; AdoMet: S-Adenosylmethionine.

Fig. 2.

HPLC analysis of blsK knockout and its complementation experiments in strain WJ2: (i) wild-type strain WJ2 as control; (ii) blsK gene knockout strain WJ2-ΔblsK (WXK3); and (iii) blsK complementation strain WJ2-ΔblsK:: blsK (WXK4).

Fig. 3.

Functional analysis of BlsK in vitro. (A) Assay with purified BlsK: (i) the LDBS standard; (ii) DBS standard; (iii) boiled BlsK as a negative control; and (iv) assay with 1 mM ATP, 10 mM leucine, and 250 μM DBS. (B) Assay of BlsK with CFE: (i) CFE of BL21 (DE3, ΔpepN)/pET44b-blsK with DBS; (ii) sample i spiked with LDBS standard; (iii) purified BlsK adding CFE of BL21 (DE3, ΔpepN) with DBS, and (iv) CFE of BL21 (DE3, ΔpepN) with DBS. In total, 1 mM ATP and 5 mM leucine were added in all groups. (C) MS/MS spectrum of the product of BlsK-catalyzed reaction (sample [B] 1): chemical structure of LDBS with the key fragmentation pattern shown. The reaction conditions were pH 8.0 and 37 °C for overnight.

Fig. 4.

The catalytic activity of BlsK requires tRNA. (A) Effect of RNase A on BlsK activity: (i) positive control of BL21 (DE3, ΔpepN) CFE with BlsK and (ii) BL21 (DE3, ΔpepN) CFE treated by RNase A with BlsK. (B) Assay of purified BlsK with tRNAs: (i) adding 1 mg/mL tRNAs to the purified BlsK reaction system and (ii) the control without tRNAs. In total, 1 mM ATP, 5 mM leucine, and 20 mM MgCl₂ were added in all reactions, pH 8.0, 37 °C overnight.

Fig. 5.

LeuRS is necessary for the activation of leucine in the BlsK-catalyzed leucyl transfer reaction: (i) Ni-NTA–purified BlsK incubated with DBS and E.coli total tRNAs; (ii) Ni-NTA–purified BlsK incubated with DBS, E.coli total tRNAs, and LeuRS; (iii) LeuRS antibody–Protein A bead–purified BlsK incubated with DBS and E.coli total tRNAs; and (iv) LeuRS antibody–treated BlsK incubated with DBS, E.coli total tRNAs, and LeuRS. In total, 1 mM ATP, and 5 mM leucine were added in all reaction mixtures. Reaction conditions: 37 °C for 1 h.

Fig. 6.

Spectroscopic characterization of recombinant BlsK. (A) Color and UV-visible spectrum analysis of BlsK protein. The blue curve represents BlsK protein and red curve is the buffer control. (B) EPR analysis of BlsK with (black) and without (blue) addition of 1 mM sodium dithionite. EPR parameters: microwave frequency, 9.39 GHz; microwave power, 2 mW; modulation amplitude, 3 Gauss; modulation frequency, 100 kHz; and temperature, 10 K. The protein concentration used was 10 mg /mL.

Fig. 7.

[3Fe-4S] is essential for the catalytic activity of BlsK. (A) HPLC analysis of BlsK-catalyzed reaction: (i) using BlsK purified under aerobic conditions; (ii) with BlsK purified under anaerobic conditions; and (iii) BlsK purified under anaerobic conditions with the [3Fe-4S] further reconstituted. (B) EDTA abolished the activity of BlsK: (i) without EDTA as control; (ii) BlsK treated with EDTA; and (iii) BlsK treated with EDTA following by the reconstitution of iron–sulfur cluster in vitro. (C) UV-visible analysis of BlsK treated with EDTA. (D) HPLC analysis of the activity of BlsK mutant proteins: (i) wild-type BlsK, and (ii–iv) represent C236S, C253S, and C259S mutant proteins, respectively. The concentration of BlsK in the reaction system was 10 μM. Enzyme assays in A and B were carried out under anaerobic conditions.