Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

Abstract

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

Tracing of Acyl Carrier Protein-channeled Mitomycin Intermediates in Streptomyces caespitosus Facilitates Characterization of the Biosynthetic Steps for AHBA–GlcN Formation and Processing

“…Inactivating mitB, mitC, mitD, or mitE exclusively led to a mutant strain that cannot produce mitomycin C (Figure S6). Different from a similar study by Kang et al, 21 we observed a trace of mitomycin C when mitF was inactivated (Figure S6). Thus, mitF is not indispensable for mitomycin biosynthesis in S. caespitosus, and its function could be partially substituted by certain NAD(P)H-dependent reductase gene(s) outside of the mit cluster.…”

“…caespitosus ATCC 27422, a strain with a product profile in which mitomycin C is dominant, we validated the involvement of mmcB (the discrete ACP protein-encoding gene) in mitomycin biosynthesis (Figure ). Clearly, mmcB is necessary, as demonstrated independently by Dairi et al and Kang et al , We then engineered mmcB to a gene coding for C-terminally 8 × His-tagged MmcB-I (Figure S2) and examined whether this variant can substitute for the function of wild-type MmcB in S. caespitosus.…”