A1 Refereed original research article in a scientific journal
ActVI-ORFA directs metabolic flux towards actinorhodin by preventing intermediate degradation
Authors: Zhu, Xuechen; Wang, Rongbin; Siitonen, Vilja; Vuksanovic, Nemanja; Silvaggi, Nicholas R.; Melançon III, Charles E.; Metsä-Ketelä, Mikko
Publisher: Public Library of Science (PLoS)
Publication year: 2024
Journal: PLoS ONE
Journal name in source: PloS one
Journal acronym: PLoS One
Article number: e0308684
Volume: 19
Issue: 8
eISSN: 1932-6203
DOI: https://doi.org/10.1371/journal.pone.0308684
Web address : https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0308684
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/457531495
The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.
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Funding information in the publication:
NIH NM-INBRE grant P20 GM103451 to CEM URL of the funder's website: https://nminbre.org/ Academy of Finland grant 354998 to MM-K URL of the funder's website: https://www.aka.fi/ The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
