Biosynthesis of the redox cofactor mycofactocin is controlled by the transcriptional regulator MftR and induced by long-chain acyl-CoA species
2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.101474
ISSN1083-351X
AutoresAigera Mendauletova, John Latham,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoMycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5′ to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA–mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization. Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc2155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5′ to mftA and found it to be highly conserved in Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, and Mycobacterium marinum. To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo, we re-engineered a fluorescent protein reporter system to express an MftA–mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc2155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization. Organic redox cofactors are essential for life. While classic flavins and nicotinamides are widely distributed across all domains of life, nature has also evolved niche cofactors in subsets of life domains. For example, in Actinobacteria, coenzyme F420 is commonly used in place of FMN in enzymes associated with carbon fixation (1Vornolt J. Kunow J. Stetter K.O. Thauer R.K. Enzymes and coenzymes of the carbon monoxide dehydrogenase pathway for autotrophic CO2 fixation in Archaeoglobus lithotrophicus and the lack of carbon monoxide dehydrogenase in the heterotrophic A. profundus.Arch. Microbiol. 1995; 163: 112-118Crossref Scopus (81) Google Scholar) and oxidation of secondary alcohols (2Aufhammer S.W. Warkentin E. Berk H. Shima S. Thauer R.K. Ermler U. Coenzyme binding in F420-dependent secondary alcohol dehydrogenase, a member of the bacterial luciferase family.Structure. 2004; 12: 361-370Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The importance of niche cofactors has long been recognized; however, detailed understanding about their biosynthesis and physiological uses has been lagging. One class of niche cofactors is derived from ribosomally synthesized and post-translationally modified peptides (RiPPs) (3Montalbán-López M. Scott T.A. Ramesh S. Rahman I.R. Van Heel A.J. Viel J.H. Bandarian V. Dittmann E. Genilloud O. Goto Y. Grande Burgos M.J. Hill C. Kim S. Koehnke J. Latham J.A. et al.New developments in RiPP discovery, enzymology and engineering.Nat. Prod. Rep. 2021; 38: 130-239Crossref PubMed Google Scholar). To achieve their mature form, the genetically encoded RiPP precursor peptide undergoes significant post-translational modifications by diverse families of tailoring enzymes (4Hetrick K.J. van der Donk W.A. Ribosomally synthesized and post-translationally modified peptide natural product discovery in the genomic era.Curr. Opin. Chem. Biol. 2017; 38: 36-44Crossref PubMed Scopus (81) Google Scholar, 5Ortega M.A. van der Donk W.A. New insights into the biosynthetic logic of ribosomally synthesized and post-translationally modified peptide natural products.Cell Chem. Biol. 2016; 23: 31-44Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 6Yang X. Van Der Donk W.A. Ribosomally synthesized and post-translationally modified peptide natural products: New insights into the role of leader and core peptides during biosynthesis.Chemistry. 2013; 19: 7662-7677Crossref PubMed Scopus (71) Google Scholar). Following synthesis by the ribosome, modifying enzymes process the precursor peptide into the mature redox cofactor. Currently, there are two known RiPP-derived redox cofactors, pyrroloquinoline quinone (7Zhu W. Klinman J.P. Biogenesis of the peptide-derived redox cofactor pyrroloquinoline quinone.Curr. Opin. Chem. Biol. 2020; 59: 93-103Crossref PubMed Scopus (9) Google Scholar), which has been well characterized, and mycofactocin (MFT), which was recently discovered. The MFT biosynthetic gene cluster (BGC) consists of mftABCDEF (Fig. 1A) and is highly conserved in mycobacteria, including pathogens, such as Mycobacterium tuberculosis (MTB), Mycobacterium ulcerans, Mycobacterium avium, and Mycobacterium bovis (8Ayikpoe R. Govindarajan V. Latham J.A. Occurrence, function, and biosynthesis of mycofactocin.Appl. Microbiol. Biotechnol. 2019; 103: 2903-2912Crossref PubMed Scopus (11) Google Scholar). As shown in Figure 1B, MFT biosynthesis starts with the MftC-catalyzed oxidative decarboxylation of the C-terminal Tyr forming MftA∗∗ (9Khaliullin B. Aggarwal P. Bubas M. Eaton G.R. Eaton S.S. Latham J.A. Mycofactocin biosynthesis: Modification of the peptide MftA by the radical S-adenosylmethionine protein MftC.FEBS Lett. 2016; 590: 2538-2548Crossref PubMed Scopus (25) Google Scholar, 10Bruender N.A. Bandarian V. The radical S-adenosyl-L-methionine enzyme MftC catalyzes an oxidative decarboxylation of the C-terminus of the MftA peptide.Biochemistry. 2016; 55: 2813-2816Crossref PubMed Scopus (40) Google Scholar) and the subsequent formation of a C–C bond resulting in a lactam derived from penultimate Val, MftA∗ (11Khaliullin B. Ayikpoe R. Tuttle M. Latham J.A. Mechanistic elucidation of the mycofactocin-biosynthetic radical-S-adenosylmethionine protein, MftC.J. Biol. Chem. 2017; 292: 13022-13033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Both reactions are dependent upon the RiPP recognition element MftB, which binds MftA and delivers it to MftC (9Khaliullin B. Aggarwal P. Bubas M. Eaton G.R. Eaton S.S. Latham J.A. Mycofactocin biosynthesis: Modification of the peptide MftA by the radical S-adenosylmethionine protein MftC.FEBS Lett. 2016; 590: 2538-2548Crossref PubMed Scopus (25) Google Scholar). Next, MftE hydrolyzes MftA∗, forming 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (12Ayikpoe R. Salazar J. Majestic B. Latham J. Mycofactocin biosynthesis proceeds through 3-amino-5-[(p-hydroxyphenyl)methyl]-4,4-dimethyl-2-pyrrolidinone (AHDP); direct observation of MftE specificity toward MftA.Biochemistry. 2018; 57: 5379-5383Crossref PubMed Scopus (12) Google Scholar). Following cleavage, MftD catalyzes the FMN-dependent oxidation of the 3-amino group, resulting in an α-keto-amide moiety within the lactam, forming premycofactocin (PMFT; Fig. 1B) (13Ayikpoe R. Latham J.A. MftD catalyzes the formation of a biologically active redox center in the biosynthesis of the ribosomally synthesized and post-translationally modified redox cofactor, mycofactocin.J. Am. Chem. Soc. 2019; 141: 13582-13591Crossref PubMed Scopus (9) Google Scholar). Finally, a recent metabolomics analysis has indicated that MftF glycosylates PMFT with up to eight β1–β4 glucans, forming mature MFT (14Peña-Ortiz L. Graça A.P. Guo H. Braga D. Köllner T.G. Regestein L. Beemelmanns C. Lackner G. Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF.Chem. Sci. 2020; 11: 5182-5190Crossref PubMed Google Scholar). In addition to mft genes, three different dehydrogenase families (TIGR03971, TIGR03989, and TIGR04266) are found in genomes only when the mft BGC is present (15Haft D.H. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners.BMC Genomics. 2011; 12: 21Crossref PubMed Scopus (63) Google Scholar). These MFT-dependent dehydrogenases have been shown to sequester NADH within their active sites and therefore require an additional electron acceptor, presumably MFT, to oxidize NADH for further catalytic turnover (16Haft D.H. Pierce P.G. Mayclin S.J. Sullivan A. Gardberg A.S. Abendroth J. Begley D.W. Phan I.Q. Staker B.L. Myler P.J. Marathias V.M. Lorimer D.D. Edwards T.E. Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors.Sci. Rep. 2017; 7: 41074Crossref PubMed Scopus (14) Google Scholar). In support of this, knockouts of the mft genes in Mycobacterium smegmatis mc2155 (Msmeg) led to the inability of the organism to maintain homeostasis of cellular NAD+/NADH pools and its inability to metabolize methanol and ethanol (17Krishnamoorthy G. Kaiser P. Lozza L. Hahnke K. Mollenkopf H. Kaufmann S.H.E. Mycofactocin is associated with ethanol metabolism in mycobacteria.mBio. 2019; 10e00190-19Crossref Scopus (11) Google Scholar, 18Dubey A.A. Jain V. Mycofactocin is essential for the establishment of methylotrophy in Mycobacterium smegmatis.Biochem. Biophys. Res. Commun. 2019; 516: 1073-1077Crossref PubMed Scopus (9) Google Scholar). The failure of the knockouts to metabolize primary alcohols is likely because of the MFT-dependent alcohol dehydrogenase, Msmeg_6242, being trapped in a reduced state in the absence of MFT. More recently, a study demonstrated that mftD, and thus MFT, is required for Mtb survival in vitro and in vivo under hypoxic conditions (19Krishnamoorthy G. Kaiser P. Constant P. Abed A. Schmid M. Brinkmann V. Daffé M. Kaufmann H.E. Role of premycofactocin synthase in growth, microaerophilic adaptation, and metabolism of Mycobacterium tuberculosis.mBio. 2021; 12e0166521Crossref Scopus (1) Google Scholar). However, until recently, direct evidence demonstrating MFT is a redox cofactor was nonexistent. This changed when it was shown that both PMFT and MFT are capable of oxidizing MFT-dependent dehydrogenases in vitro (13Ayikpoe R. Latham J.A. MftD catalyzes the formation of a biologically active redox center in the biosynthesis of the ribosomally synthesized and post-translationally modified redox cofactor, mycofactocin.J. Am. Chem. Soc. 2019; 141: 13582-13591Crossref PubMed Scopus (9) Google Scholar, 14Peña-Ortiz L. Graça A.P. Guo H. Braga D. Köllner T.G. Regestein L. Beemelmanns C. Lackner G. Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF.Chem. Sci. 2020; 11: 5182-5190Crossref PubMed Google Scholar). Despite knowing the structure, biosynthesis, and redox attributes of MFT, information about physiological processes that require MFT has been lagging. One way to address the physiological dependence on MFT is to understand how MFT biosynthesis is regulated. Currently, it is thought that putative TetR-like protein MftR is a regulator of MFT biosynthesis (20Cuthbertson L. Nodwell J.R. The TetR family of regulators.Microbiol. Mol. Biol. Rev. 2013; 77: 440-475Crossref PubMed Scopus (280) Google Scholar, 21Minch K.J. Rustad T.R. Peterson E.J.R. Winkler J. Reiss D.J. Ma S. Hickey M. Brabant W. Morrison B. Turkarslan S. Mawhinney C. Galagan J.E. Price N.D. Baliga N.S. Sherman D.R. The DNA-binding network of Mycobacterium tuberculosis.Nat. Commun. 2015; 6: 5829Crossref PubMed Scopus (128) Google Scholar). In general, TetR family regulators (TFRs) are transcription repressors and implicated in the regulation of efflux pumps (22Grkovic S. 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Horinouchi S. Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.J. Bacteriol. 1995; 177: 6083-6092Crossref PubMed Google Scholar), depending on the system. MftR regulation of the mft BGC is supported by bioinformatics, which suggests that the gene proximity of mftR and its arrangement to the mft BGC is indicative of regulatory control of MFT biosynthesis (35Ahn S.K. Cuthbertson L. Nodwell J.R. Genome context as a predictive tool for identifying regulatory targets of the TetR family transcriptional regulators.PLoS One. 2012; 7e50562Crossref Scopus (45) Google Scholar). In addition, a transcriptomics study of macrophage samples infected with MTB showed that upregulation of the MftR homolog, Rv0691c, led to the repression of mftB, mftC, and mftD (36Peterson E.J. Bailo R. Rothchild A.C. Arrieta-Ortiz M.L. Kaur A. Pan M. Mai D. Abidi A.A. Cooper C. Aderem A. Bhatt A. Baliga N.S. Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation.Mol. Syst. Biol. 2019; 15e8584Crossref Scopus (28) Google Scholar). Currently, the specific DNA operator sequence that MftR recognizes, its regulatory role over the mft BGC in Msmeg, and the conditions that MftR could regulate MFT biosynthesis are unknown. Here, we report that msmeg_1420, annotated as MftR, is a transcriptional repressor of the mft BGC in Msmeg. We found that MftR binds a DNA sequence in the promoter region of the mft BGC. We mapped the 27 bp mft operator (Omft) by DNase I footprinting and measured dissociation constant (Kd) of the MftR–Omft complex by fluorescence anisotropy. We employed relative RT–quantitative PCR (qPCR) to demonstrate that overexpression of MftR results in the repression of mft genes in Msmeg. To determine under what conditions the biosynthesis of MFT might be induced, we employed EMSAs and isothermal titration calorimetry (ITC) to identify effectors of MftR. To demonstrate that identified effectors translate in vivo, we use an engineered fluorescence reporter system to show that effectors supplemented to growth media induces the expression of the mft BGC in Msmeg and quantify the induction relative to RT–qPCR. These findings provide a mechanism to understand the physiological conditions that MFT is regulated and thus required in vivo. To provide evidence that MftR is a regulator of MFT biosynthesis, we ran a series of EMSAs to demonstrate that MftR binds to the MFT promoter region. To begin with, recombinant His-tagged Msmeg MftR was purified from Escherichia coli (Fig. S1) and the 565 bp promoter region (Pmft) between −470 to +95 relative to MftA was PCR amplified. Next, EMSAs were carried out in triplicate with a fixed concentration of the PCR-amplified Pmft and varying concentrations of MftR. As shown in Figure 2A, the addition of MftR to unlabeled Pmft resulted in a single impeded band in a concentration-dependent manner with an estimated Kd ∼ 0.6 μM. This result indicates that at least a single binding site of MftR with at least one binding affinity is present in the Pmft regulatory region. To determine the exact location of the MftR-binding site in the mft regulatory region, DNase I protection assays were performed using a Pmft DNA probe labeled with 6-carboxyfluorescein (6-FAM), in the presence and absence of MftR. As shown in Figure 2B, MftR protected a single region extending from −79 to −53 from DNase I digestion. The shift in DNase I hypersensitivity by three nucleotides when MftR is present suggests the establishment of new contacts being made to and/or a modification of the DNA structure. To validate this finding, the MftR protected sequence was synthesized with a 6-FAM label and used in a subsequent EMSA. As shown in Figure 2C, increasing concentrations of MftR resulted in a single concentration-dependent impeded band, consistent with the original EMSA with Pmft. Controls with a "cold" competitive ligand and with a nonspecific DNA sequence (Fig. S2) suggest that the interaction between MftR and the identified region is specific. In addition, fluorescence anisotropy experiments were carried out to estimate the binding affinity between MftR and the 6-FAM-labeled 27 bp sequence. Consistent with EMSAs, increasing concentrations of MftR resulted in a concentration-dependent change in polarization, which upon fitting three independent experiments to a single-site binding model, led to an observed Kd of 1.3 ± 0.6 μM (Fig. 2D). As a result, we propose that the MFT operator (Omft) sequence includes at least one MftR binding motif within the sequence 5′-TCCATTCTGGCACTCGATGCCATATAT (Fig. 2E). Next, we employed real-time qRT–PCR analysis to demonstrate that MftR regulates the mft BGC in vivo. We measured the transcript levels of mftA-F and mftR in wildtype Msmeg and compared them to the transcript levels in Msmeg harboring a mycobacterial expression vector consisting of mftR under the control of the constitutive expression promoter Psmyc (pMftR+). Overproduction of mftR in the expression strain, as compared with wildtype Msmeg, was confirmed by qRT–PCR analysis, which revealed that the mftR transcript abundance was increased by approximately fivefold (Fig. 3). Conversely, overproduction of mftR led to reduced transcript levels in all mft biosynthetic genes. Notably, the transcript levels of mftA and mftC were reduced by ∼15-fold and ∼20-fold, respectively. However, the most remarkable change in transcript levels was that of mftD, which was reduced nearly 80-fold. The transcript levels for mftB, mftE, and mftF were also decreased however, to a lesser extent ( 84%. As shown in the WebLogo (37Crooks G.E. Hon G. Chandonia J. Brenner S.E. WebLogo : A sequence logo generator.Genome Res. 2004; 14: 1188-1190Crossref PubMed Scopus (7851) Google Scholar) depiction of the multiple sequence alignment of all sequences (Fig. 2E), we found that putative Omft regions are highly conserved. In addition, our analysis identified a palindromic region consisting of the residues T-N2-GGCA-N5-TGCC-N2-A. Despite the apparent conservation of the palindrome, single nucleotide replacements within the sequence did not impede the ability of MftR to bind Omft in fluorescence polarization (FP) or EMSAs (data not shown). Next, we assessed which metabolites activate MftR and thus could induce MFT production. To do so, we carried out competitive EMSAs where FAM-labeled Omft and MftR were incubated in the presence of potential effectors. We initially chose our effectors based on cholesterol catabolism, a process that putatively includes MFT biosynthetic genes (38Pandey A.K. Sassetti C.M. Mycobacterial persistence requires the utilization of host cholesterol.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (672) Google Scholar). Despite the loose association of MFT to cholesterol catabolism, we did not observe DNA release by MftR in the presence of cholesterol (Fig. 4A, lane 3), propionyl-CoA (Fig. 4A, lane 6), succinyl-CoA (not shown), and acetoacetyl-CoA (not shown). Knowing that TFRs have a propensity to be activated by fatty acyl-CoAs (20Cuthbertson L. Nodwell J.R. The TetR family of regulators.Microbiol. Mol. Biol. Rev. 2013; 77: 440-475Crossref PubMed Scopus (280) Google Scholar), we expanded our effector screening to include short-chain, medium-chain, and long-chain acyl-CoAs. Subsequently, we observed that the addition of myristoyl-CoAs and oleoyl-CoAs disrupted the MftR–Omft complex (Fig. 4A, lanes 8 and 9) and resulted in both bound and unbound Omft. Conversely, the addition of fatty acid carboxylates did not result in the same disruption of the MftR–Omft complex (Fig. 4A, lane 10), suggesting that CoA is a requisite for acyl-CoA binding. However, CoA alone did not disrupt the MftR–Omft complex either (Fig. 4A, lane 4), suggesting that protein contacts with the ligand rely on both the fatty acid and the CoA. ITC experiments were performed to validate the competitive EMSA findings and to determine the specificity and affinity of MftR toward acyl-CoAs. A typical thermogram was obtained when oleoyl-CoA was titrated into MftR (Fig. 4B). The Kd value (1.4 ± 0.1 μM; Fig. 4C) and the number of binding sites (∼0.6 sites per monomer MftR) were obtained from the nonlinear one-site model to the normalized fitting curve. The Kd value is comparable to known mycobacterial TFRs that are activated by oleoyl-CoA (39Lara J. Diacovich L. Trajtenberg F. Larrieux N. Malchiodi E.L. Fernández M.M. Gago G. Gramajo H. Buschiazzo A. Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine.Nat. Commun. 2020; 11: 1-13Crossref PubMed Scopus (9) Google Scholar, 40Dong W. Nie X. Zhu H. Liu Q. Shi K. You L. Zhang Y. Fan H. Yan B. Niu C. Lyu L.D. Zhao G.P. Yang C. Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2019305118Crossref Scopus (5) Google Scholar). To establish the specific acyl-CoA(s) that activate MftR, we measured the Kds for myristoyl-CoA, palmitoyl-CoA, and steroyl-CoA and found the values to be within the 2 to 4 μM range (Fig. 4C). Of note, we observed a ∼10-fold decrease in binding affinity with lauroyl-CoA as compared with oleoyl-CoA. This drop in affinity is consistent with EMSAs that indicated medium-chain and short-chain acyl-CoAs do not disrupt the MftR–Omft complex. Taken together with the EMSAs, our ITC data suggest that MftR, and thus likely MFT biosynthesis, is activated by long-chain acyl-CoAs. Next, we examined which amino acid residues contribute to the interaction between MftR and oleoyl-CoA. The crystal structure of Msmeg MftR is currently unavailable; however, an unpublished structure of Rhodococcus jostii RHA1 MftR has been deposited in the Protein Data Bank (PDB) (PDB ID: 2RAE, 54% identical, Fig. S3). Using this structure, we employed SwissDock to model oleoyl-CoA bound to MftR (Fig. 5) (41Grosdidier A. Zoete V. Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS.Nucleic Acids Res. 2011; 39: W270-W277Crossref PubMed Scopus (1001) Google Scholar, 42Grosdidier A. Zoete V. Michielin O. Fast docking using the CHARMM force field with EADock DSS.J. Comput. Chem. 2011; 32: 2149-2159Crossref PubMed Scopus (283) Google Scholar). From the docked structure, we identified eight conserved residues on MftR that were expected to create the acyl-binding pocket or bind CoA through electrostatic interactions. Following single amino acid replacements of the residues, we measured the Kd values of the mutant proteins to oleoyl-CoA using ITC. For the putative acyl-binding pocket residues Phe65, Phe96, and Ile114, mutations to alanine led to no or modest change to the Kd for oleoyl-CoA (Table 1). This is consistent with other TFR proteins where single-residue changes to the acyl-binding pocket resulted in little to no change in their Kd to acyl-CoAs (39Lara J. Diacovich L. Trajtenberg F. Larrieux N. Malchiodi E.L. Fernández M.M. Gago G. Gramajo H. Buschiazzo A. Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine.Nat. Commun. 2020; 11: 1-13Crossref PubMed Scopus (9) Google Scholar). However, we cannot rule out the possibility that Phe65, Phe96, and Ile114 do not participate in acyl-CoA binding. Conversely, when His68 was mutated to alanine, binding of oleoyl-CoA by the protein was undetectable. To validate this observation, competitive EMSAs were carried out using the H68A mutant. Here, it was observed that the H68A mutant remained bound to Omft even in the presence of 100 μM oleoyl-CoA (Fig. S4A). Thus, it is highly likely that His68 is an important residue for oleoyl-CoA binding. To evaluate residues that were expected to interact with CoA, Gln15, Asp16, Ser67, and Asp66 were targeted. Of these, mutants of Asp16 and Ser67 had the greatest effect (Table 1). For instance, the S67A mutation led to a fivefold increase in the Kd value for ole
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