Fig. 4: LimB catalyzes post-crotonylation oxidation on Acs. | Communications Biology

Fig. 4: LimB catalyzes post-crotonylation oxidation on Acs.

From: Post-crotonylation oxidation by a monooxygenase promotes acetyl-CoA synthetase degradation in Streptomyces roseosporus

Fig. 4

a. Bacterial two-hybrid assays for the interactions of LimB and acyl-CoA synthetases. The E. coli BTH101 containing LimB (T18-LimB) or the void vector T18 along with other acyl-CoA synthetases (Orf1034, Orf1035, Orf3275, Orf5336, Orf5795) were patched on the LB medium, grown for 3 days and photographed. b In vitro oxidation assays with purified His-tagged LimB, LimB-N and Acs. Acs was incubated LimB (lanes 3 and 4) or LimB-N (lanes 5 and 6), coenzymes and Western blot assays were demonstrated with the anti-crotonylation (α-Kcr) antibody for the crotonylation level and the anti-His (α-His) antibody for the loading control of Acs. The control assays without LimB/LimB-N or coenzymes (NADH, FAD) or neither of them were also included (lane 1-3 and 5). c In vivo crotonylation assays of Acs after limB deletion. Acs was expressed with a 3×FLAG tag in S. roseosporus, and immuno-purified from the lysate of WT and the ΔlimB mutant. Western blot assays were demonstrated with α-Kcr antibody for the crotonylation level and α-FLAG antibody for the loading control of Acs. d Bacterial two-hybrid assays for the interaction of LimB N-terminal domain (LimB-N) and Orf3275 (Acs). The E. coli BTH101 containing LimB-N or the void vector T18 along with Acs or the vector T25, respectively, were patched on the LB medium, grown for 3 days and photographed.

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