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

  1. Gajanan S 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

  • Curated by eLife

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    Evaluation Summary:

    This study addresses the structural basis of the ability of fatty acyl-AMP ligases (FAAL) to exclude condensation of activated fatty acids with coenzyme-A and facilitate the reaction with other 4-phosphopantetheine linked acceptors. This issue is of significant interest with regard to understanding how certain fatty acids are channeled to specific metabolic fates. The structural question at hand is the apparent discrimination of the CoA moiety (adenosine 3',5'-bisphosphate) versus a holo-ACP tethered to the 4-phosphopantethein head group. This work will contribute significantly to our current knowledge of how distinct classes of enzymes divert fatty acids to virulent lipids in mycobacteria, and it will be more broadly of interest for metabolic engineering.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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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 that 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 organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

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  1. Version published to 10.7554/elife.70067 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In this manuscript the authors have addressed the structural reasons for the ability of fatty acyl-AMP ligases (FAAL) to exclude condensation of activated fatty acids with coenzyme-A and facilitate the reaction with other 4-phosphopantetheine linked acceptors. This issue is of significant interest with regard to understanding how certain fatty acids are channeled to specific metabolic fates. The structural issue is the apparent discrimination of the CoA moiety (adenosine 3',5'-bisphosphate) versus a holo-ACP tethered to the 4-phosphopantethein head group. The authors identified a number of probable issues between FAAL and FACL enzymes.

    1. The authors have shown that many of the FAAL enzymes lack the positively charged residues that have been shown previously to function in recognition of the CoA moiety (Figure 1a).

    Thank you for highlighting one of the strong reasons, “lack of positive selection”, as to why FAALs do not bind CoA.

    1. They have highlighted a number of residues within the putative binding site for the 4'-pantatheine moieties in the FAAL enzymes that likely preclude the binding of this portion of the substrate. They have subsequently mutated these residues in FAAL enzymes from three different organisms and have shown in certain instances that the mutated enzymes are now able to functionally activate CoA (Figure 2c). The authors, however, should have attempted to explain why some of the FAALs behaved differently than others. For example, why does the F284A/M233A mutant of MsFAAL32 function so differently than the corresponding mutant of RsFAAL? Also, the residue numbering is confusing to this reviewer. Thus, in Figures 1b and F1c the specific methionine and phenylalanine residues that are highlighted are labeled as M231 and F279, respectively. Yet in Figure 2 the methionine mutated is listed as M227 and the phenylalanine is listed as F275. Why is there a residue difference of 4?

    We thank the reviewer for raising this interesting point about the differential activity. Variable residues lining the CoA-binding site can influence the reaction in unpredictable ways, which possibly explains the observed differential activity. A detailed analysis has been presented as an answer to the essential review question 1 and a short description is added at page-12.

    We thank the reviewer for pointing out the issue with erroneous residue numbering of EcFAAL. The difference in residues is a typographical error and has been corrected in the figure.

    1. The authors have provided further support for the inability of the apparent canonical site in the FAAL enzymes to be functional by mutating the residues within the active sites of certain FACL enzymes to the bulkier ones found in the FAAL enzymes. Many of the constructs resulted in the loss of function and their ability to activate CoA. However, the loss of function was not uniform across the three FACL enzymes chosen, and the authors have done an insufficient job of explaining the differences. For example, the A276F/A232M mutant of AfFACL is devoid of CoA activity but the corresponding mutants of MtFACL13 and EcFACL are fully functional.

    As previously explained in the answer to question-2, multiple highly divergent residues lining the CoA-binding site in FACLs possibly explains the observed differential activity. A detailed analysis as an answer to the essential review question 2 has been presented and a short description is added at page-12.

    1. The authors have identified a putative alternative binding site for the 4'-pantatheine moiety using various computational searches that can apparently distinguish between CoA and holo-ACP (Figure 3). Mutations of residue within this newly identified pocket (Figure 4c) significantly diminishes the condensation with the ACP from E. coli.

    We thank you for highlighting the identification of the alternative site and its validation through structure-guided mutagenesis and biochemistry.

    Reviewer #2 (Public Review):

    This manuscript succeeds in experimentally establishing the rationale for a acyl-carrier protein (ACP) substrate specificity of the fatty acyl-AMP ligases. While these enzymes structurally resemble the CoA-dependent fatty acyl CoA-ligases (FACLs), the authors demonstrate that the FAALs use a novel binding site to accommodate the ACP substrate. The biochemical studies are solid and clear cut but the evolutionary analysis could be bolstered with additional bioinformatics analysis. With said analysis, this manuscript would contribute significantly to our current knowledge of distinct classes of enzymes that divert fatty acids to virulent lipids in mycobacteria.

    We appreciate the reviewer’s suggestion to include genomic neighborhoods of PKS/NRPS showing the presence of FAALs to strengthen the evolutionary analysis. A detailed sequence analysis has been performed and presented as answer to essential review question 3. The tabulation of the analysis is now presented as Supplementary table-III.

    Reviewer #3 (Public Review):

    This study that attempted to understand how an important family of enzymes (FAALs) involved in fatty acid activation and transfer can recognise one form or a substrate in preference to another. They were trying to identify how certain enzymes recognise the 4'-PP-SH arm of CoASH is not recognised but the same arm attached to a small acyl carrier protein (ACP) is preferred. The author used detailed analysis of crystal structures in the PDB of many examples of ligand-bound ANLs, sequence analysis and molecular modelling to direct site directed mutagenesis of the FAALs and reveal important elements in the enzyme involved in substrate recognition and discriminaton. This is a major strength of the paper. e.g. SFig. 7. There is also a nice evolution discussion about the origin of the ANL family.

    We thank the reviewer for highlighting the important mechanistic aspects that enable CoA discrimination in FAALs and their evolutionary conservation, which led to the proposition of parallel evolution of FAALs and FACLs.

    Once they have identified potential residues involved in CoSH or ACP-SH (holo) binding they make a number of mutants of various enzymes. These included EcFAAL, MsFAAL32, RsFAAL, MxFAAL, AfFACL etc. They appear to use a radioactive substrate assay (using hot FA) and measure incorporation with various gels which are scanned and counted. This is where I began to get lost and to me it is a major weakness. They compare WT with mutants including site directed mutants (made deletions of deltaFS1 etc) . I found Fig. 2 confusing. The gels are also confusing.

    We appreciate the reviewers’ suggestion to improve the description on how the experiment was performed along with the readouts in the form of gels or TLCs. The experimental section has been elaborated detailing different steps of the biochemical assays and provided as answer to essential review question 6. We have modified the legends of figure-2 and supplementary figure-6 labelled the figures with relevant information to make things clearer. We hope that these changes should address the concerns of the reviewer.

    There appears to be a problem with the apo- to holo-ACP conversion - why didn't Bs Sfp work to 100%? There is one mass spec analysis - why wasn't more used?

    We agree with the reviewers’ comment that apo- to holo- conversion was not complete as expected. Initially, we assumed complete conversion to holo-ACP form by BsSfp and used it for biochemical analysis using traditional CS-Urea PAGE, however it failed to show any differential migration. We then resorted to modifying experimental conditions to improve the holo-conversion including cloning the BsSfp afresh and further check its efficiency using Coomassie stained CS-Urea-PAGE. It was only after we used MALDI-TOF, we realized that at least the only ACP that could be detected had ~50-60% conversion. As the remaining ACPs failed to fly and get detected, we have not presented the analysis for the remaining. Despite extensive efforts, we could not see any improvement in the conversion process. So, the reduced conversion efficiency appears to be a protein specific phenomenon and need further investigation.

    The reaction is not clearly drawn to begin with - I work in this area. Be clear in the steps. Draw out the FA + MgATP to give acyl-adenylate + PPi. Then, add CoASH or ACP-SH and show formation of acyl-CoA + AMP or acyl-ACP + AMP.

    We thank the reviewer for the suggestion and have now included the reaction schematic as supplementary figure-1b.

    There are many other assays to measure activity e.g. PPi coupled or measure AMP formation to back up data from the radioactivity.

    We thank the reviewer for the suggestion, and we take this the opportunity to explain our choice of methodology for assessing the biochemical reactions. The coupled assays typically measure the PPi release or AMP, which in turn rely on other enzyme systems and are excellent means to assess the first step of the reaction directly. However, it is an indirect approach to use these for measuring the second step of the reaction. Particularly, in instances where we need to quantify the efficiency of the second step mutants, which are not affected in the first step, it cannot be ascertained using how much PPi or AMP is formed. We agree that mass-spectrometry is another approach for these systems but frequent access on a continuous basis was not feasible. Therefore, we chose an approach that allows us to see the products, acyl-AMP and acyl-CoA/acyl-ACP, directly via a TLC or Urea-PAGE. The potential hazard to this approach of course is the usage of radioactivity.

    Figure 4 confuses me a lot. As does Supp Fig 6.

    The legends of figure-4 and Supplementary figure-6 are modified, and the figures appropriately labelled to include more information. We hope that these changes should address the concerns of the reviewer.

    It appears the authors are looking for a yes/no answer - active with CoASH or ACP-SH. A Table would help to summarise.

    We thank the reviewer for the suggestion and have now tabulated the results of gain of function and loss of function in FAALs and FACLs, respectively, as a supplementary table-V.

  3. eLife

    Evaluation Summary:

    This study addresses the structural basis of the ability of fatty acyl-AMP ligases (FAAL) to exclude condensation of activated fatty acids with coenzyme-A and facilitate the reaction with other 4-phosphopantetheine linked acceptors. This issue is of significant interest with regard to understanding how certain fatty acids are channeled to specific metabolic fates. The structural question at hand is the apparent discrimination of the CoA moiety (adenosine 3',5'-bisphosphate) versus a holo-ACP tethered to the 4-phosphopantethein head group. This work will contribute significantly to our current knowledge of how distinct classes of enzymes divert fatty acids to virulent lipids in mycobacteria, and it will be more broadly of interest for metabolic engineering.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript the authors have addressed the structural reasons for the ability of fatty acyl-AMP ligases (FAAL) to exclude condensation of activated fatty acids with coenzyme-A and facilitate the reaction with other 4-phosphopantetheine linked acceptors. This issue is of significant interest with regard to understanding how certain fatty acids are channeled to specific metabolic fates. The structural issue is the apparent discrimination of the CoA moiety (adenosine 3',5'-bisphosphate) versus a holo-ACP tethered to the 4-phosphopantethein head group. The authors identified a number of probable issues between FAAL and FACL enzymes.

    1. The authors have shown that many of the FAAL enzymes lack the positively charged residues that have been shown previously to function in recognition of the CoA moiety (Figure 1a).

    2. They have highlighted a number of residues within the putative binding site for the 4'-pantatheine moieties in the FAAL enzymes that likely preclude the binding of this portion of the substrate. They have subsequently mutated these residues in FAAL enzymes from three different organisms and have shown in certain instances that the mutated enzymes are now able to functionally activate CoA (Figure 2c). The authors, however, should have attempted to explain why some of the FAALs behaved differently than others. For example, why does the F284A/M233A mutant of MsFAAL32 function so differently than the corresponding mutant of RsFAAL? Also, the residue numbering is confusing to this reviewer. Thus, in Figures 1b and F1c the specific methionine and phenylalanine residues that are highlighted are labeled as M231 and F279, respectively. Yet in Figure 2 the methionine mutated is listed as M227 and the phenylalanine is listed as F275. Why is there a residue difference of 4?

    3. The authors have provided further support for the inability of the apparent canonical site in the FAAL enzymes to be functional by mutating the residues within the active sites of certain FACL enzymes to the bulkier ones found in the FAAL enzymes. Many of the constructs resulted in the loss of function and their ability to activate CoA. However, the loss of function was not uniform across the three FACL enzymes chosen, and the authors have done an insufficient job of explaining the differences. For example, the A276F/A232M mutant of AfFACL is devoid of CoA activity but the corresponding mutants of MtFACL13 and EcFACL are fully functional.

    4. The authors have identified a putative alternative binding site for the 4'-pantatheine moiety using various computational searches that can apparently distinguish between CoA and holo-ACP (Figure 3). Mutations of residue within this newly identified pocket (Figure 4c) significantly diminishes the condensation with the ACP from E. coli.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript succeeds in experimentally establishing the rationale for a acyl-carrier protein (ACP) substrate specificity of the fatty acyl-AMP ligases. While these enzymes structurally resemble the CoA-dependent fatty acyl CoA-ligases (FACLs), the authors demonstrate that the FAALs use a novel binding site to accommodate the ACP substrate. The biochemical studies are solid and clear cut but the evolutionary analysis could be bolstered with additional bioinformatics analysis. With said analysis, this manuscript would contribute significantly to our current knowledge of distinct classes of enzymes that divert fatty acids to virulent lipids in mycobacteria.

  6. eLife

    Reviewer #3 (Public Review):

    This study that attempted to understand how an important family of enzymes (FAALs) involved in fatty acid activation and transfer can recognise one form or a substrate in preference to another. They were trying to identify how certain enzymes recognise the 4'-PP-SH arm of CoASH is not recognised but the same arm attached to a small acyl carrier protein (ACP) is preferred.
    The author used detailed analysis of crystal structures in the PDB of many examples of ligand-bound ANLs, sequence analysis and molecular modelling to direct site directed mutagenesis of the FAALs and reveal important elements in the enzyme involved in substrate recognition and discriminaton. This is a major strength of the paper. e.g. SFig. 7. There is also a nice evolution discussion about the origin of the ANL family.

    Once they have identified potential residues involved in CoSH or ACP-SH (holo) binding they make a number of mutants of various enzymes.
    These included EcFAAL, MsFAAL32, RsFAAL, MxFAAL, AfFACL etc. They appear to use a radioactive substrate assay (using hot FA) and measure incorporation with various gels which are scanned and counted. This is where I began to get lost and to me it is a major weakness. They compare WT with mutants including site directed mutants (made deletions of deltaFS1 etc) .
    I found Fig. 2 confusing.
    The gels are also confusing.
    There appears to be a problem with the apo- to holo-ACP conversion - why didn't Bs Sfp work to 100%?
    There is one mass spec analysis - why wasn't more used?

    The reaction is not clearly drawn to begin with - I work in this area.
    Be clear in the steps.
    Draw out the FA + MgATP to give acyl-adenylate + PPi. Then, add CoASH or ACP-SH and show formation of acyl-CoA + AMP or acyl-ACP + AMP.
    There are many other assays to measure activity e.g. PPi coupled or measure AMP formation to back up data from the radioactivity.
    Figure 4 confuses me a lot. As does Supp Fig 6.

    It appears the authors are looking for a yes/no answer - active with CoASH or ACP-SH.
    A Table would help to summarise.

  7. Version published to 10.1101/2021.05.04.442589v1 on bioRxiv