A universal pocket in Fatty acyl-AMP ligases ensures redirection of fatty acid pool away from Coenzyme A-based activation

Abstract

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3', 5'-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organisations. The universal distribution of FAALs suggests they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, Figure 4, Figure supplement 5b and Figure supplement 6.

Article and author information

Author details

  1. Gajanan Shrikant Patil

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  2. Priyadarshan Kinatukara

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2210-2369
  3. Sudipta Mondal

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3923-7449
  4. Sakshi Shambhavi

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8852-1542
  5. Ketan D Patel

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4254-3145
  6. Surabhi Pramanik

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  7. Noopur Dubey

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  8. Subhash Narasimhan

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  9. Murali Krishna Madduri

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  10. Biswajit Pal

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    No competing interests declared.
  11. Rajesh S Gokhale

    National Institute of Immunology, New Delhi, India
    Competing interests
    No competing interests declared.
  12. Rajan Sankaranarayanan

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    For correspondence
    sankar@ccmb.res.in
    Competing interests
    Rajan Sankaranarayanan, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4524-9953

Funding

Department of Biotechnology, Ministry of Science and Technology, India

  • Gajanan Shrikant Patil

Council of Scientific and Industrial Research, Ministry of Science and Technology, India

  • Sudipta Mondal

University Grants Commission

  • Sakshi Shambhavi

Council of Scientific and Industrial Research, Ministry of Science and Technology, India

  • Rajan Sankaranarayanan

Science and Engineering Research Board, India

  • Rajan Sankaranarayanan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Frank Raushel, Texas A&M University, United States

Publication history

  1. Preprint posted: May 4, 2021 (view preprint)
  2. Received: May 5, 2021
  3. Accepted: September 6, 2021
  4. Accepted Manuscript published: September 7, 2021 (version 1)
  5. Version of Record published: September 23, 2021 (version 2)

Copyright

© 2021, Patil et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,956
    Page views
  • 221
    Downloads
  • 2
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Gajanan Shrikant Patil
  2. Priyadarshan Kinatukara
  3. Sudipta Mondal
  4. Sakshi Shambhavi
  5. Ketan D Patel
  6. Surabhi Pramanik
  7. Noopur Dubey
  8. Subhash Narasimhan
  9. Murali Krishna Madduri
  10. Biswajit Pal
  11. Rajesh S Gokhale
  12. Rajan Sankaranarayanan
(2021)
A universal pocket in Fatty acyl-AMP ligases ensures redirection of fatty acid pool away from Coenzyme A-based activation
eLife 10:e70067.
https://doi.org/10.7554/eLife.70067

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Hwan Bae et al.
    Research Advance

    Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Sarah R Hansen et al.
    Research Article

    In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used co-localization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.