1. Biochemistry and Chemical Biology

Biochemical basis for the regulation of biosynthesis of antiparasitics by bacterial hormones

  1. Iti Kapoor
  2. Philip Olivares
  3. Satish K Nair  Is a corresponding author
  1. Department of Biochemistry, University of Illinois at Urbana Champaign, United States
  2. Institute for Genomic Biology, University of Illinois at Urbana Champaign, United States
  3. Center for Biophysics and Computational Biology, University of Illinois at Urbana Champaign, United States
https://doi.org/10.7554/eLife.57824
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  1. Iti Kapoor
  2. Philip Olivares
  3. Satish K Nair
(2020)
Biochemical basis for the regulation of biosynthesis of antiparasitics by bacterial hormones
eLife 9:e57824.
https://doi.org/10.7554/eLife.57824
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18 figures, 3 tables and 2 additional files

Figures

Figure 1
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Chemical structures and retrosynthetic scheme for avenolide.

(A) Representation of the mechanism for hormone-induced transcriptional activation in bacteria. (B) Structures of representative compounds from the four known classes of bacterial hormones. A-factor is a γ-butyrolactone, avenolide is an alkylbutenolide, SRB1 is a 2-alkyl-3-methyl-4-hydroxybutenolide, and MMF1 is a 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid. (C) Retrosynthetic scheme for avenolide synthesis involving five key reactions. (D) Overall summarized and synthetic scheme for total synthesis of (4S,10R)-avenolide with total number of steps and reaction yields.

Figure 2
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Structural characterization of the AvaR1-avenoide binding interaction.

(A) Structure of the AvaR1 homodimer in the absence of bound ligand. One monomer is shown colored in blue and another in brown. (B) Co-crystal structure of one monomer of AvaR1 (in pink) bound to (4S,10R)-avenolide (in yellow ball-and-stick). The ligand-binding domain (LBD) and the DNA-binding domain (DBD) are indicated. (C) Difference Fourier map (countered at 3 σ) calculated with coefficients |F(obs)|–|F(calc)| with the coordinates of the avenolide omitted prior to one round of refinement. The coordinates of the final structure are superimposed. (D) Superposition of the structures of the DBD of AvaR1 in the presence (brown) and absence (cyan) of bound ligand. Ligand binding induces a 10o shift in this domain that would preclude DNA binding. (E) Representative binding isotherm for the interaction of AvaR1 with (4S,10R)-avenolide indicative of a 1:1 binding stoichiometry.

Figure 3
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Close-up views of AvaR1-ligand and DNA structures.

(A) Multiple sequence alignment of various GBL-like receptors for which ligand specificity is known. The color-coding of the receptor names reflects the ligand class as colored in Figure 1B. Residues involved in interactions with the lactone are marked by green triangles, those interacting with the alkyl chain are marked by red diamonds, and those proposed to be involved in mediating hormone-dependent conformational movement are marked with orange circles. (B) Close-up view of the hormone-binding cavity showing residues that are in contact with the bound ligand. (C) Spatial orientation of conserved residues that are proposed to induce movement of the DBD in response to binding of the hormone at the ligand-binding domain. (D) Close-up view of the DBD of AvaR1 in complex with the aco ARE.

Figure 4
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Sequence Similarity Networks of likely butenolide gene clusters.

(A) Sequence Similarity Networks (SSN) showing the relationship between different clades of putative butenolide receptors. Characterized receptors are shown in light green. (B) Conservation of sequences amongst the 90 putative butenolide receptors identified by bioinformatics mapped onto the structure of AvaR1. The color range indicates the least conserved (cyan) through to the most conserved (purple). (C) Genomic synteny used to cull sequences for the SSN.

Chemical structure 1
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One step synthesis of (R)-2-Methylbutane-1,2-diol (1) from 2-methyl-1-butene via Sharpless asymmetric dihydroxylation.
Chemical structure 2
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Single step synthesis of allyl alcohol 17 from epoxy 10.
Appendix 1—chemical structure 1
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Chemical structures and synthetic scheme for intermediate 9.
Appendix 1—chemical structure 2
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Chemical structures and synthetic scheme for avenolide.
Appendix 1—figure 1
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Proposed biosynthetic pathway for avenolide and putative roles of biosynthetic genes.
Appendix 1—figure 2
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Crystallographic studies with DNA oligonucleotides.

(a) AvaR1 binding site in the upstream region of aco gene. (b) Crystals obtained with DNA Oligo Pal2-1 variant sequences; below the structure on the right is the screenshot from COOT depicting the electron density of the DNA oligo. (c) Proposed sequences containing linker with 1 bp or 2bp length.

Appendix 1—figure 3
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1H and 13C NMR spectra of key intermediates and final product (4S,10R)-avenolide.
Appendix 1—figure 4
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1H and 13C NMR spectra of (R)-2-Methylbutane-1,2-diol (1).
Appendix 1—figure 5
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1H and 13C NMR spectra of 5-(tert-Butyldiphenylsilanyloxy)-pentanal (4).
Appendix 1—figure 6
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1H and 13C NMR spectra of (E)-7-((tert-butyldiphenylsilyl)oxy)hept-2-en-1-ol (6).
Appendix 1—figure 7
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1H and13C NMR spectra of iodo-alkene intermediate; (E)-2-((7-iodohept-2-en-1-yl)oxy)tetrahydro-2H-pyran (11).
Appendix 1—figure 8
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1H and 13C NMR spectra of allyl alcohol intermediate; (R)-8-((2S,3S)3-(Hydroxymethyl)oxiran-2-yl)3-((4-methoxybenzyl)oxy)3-methyloctan-4-one (17).
Appendix 1—figure 9
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1H and 13C NMR spectra of acrylyl alkene intermediate; (3S,9R)-9-((4-methoxybenzyl)oxy)-9-methyl-8-oxoundec-1-en-3-yl acrylate (18).
Appendix 1—figure 10
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1H and 13C NMR spectra of final product; (4S,10R)-avenolide (13).

Tables

Appendix 1—table 1
List of all of the acoARE oligonucleotides tried for co-crystallization with AvaR1.

Pal in the name denotes the palindromic sequences that have been designed using the first or the second half of the symmetric sequence, which are self-annealing. The first half of the sequence is complementary to the second half.

Target sequence (acoARE): 5′-CTTGAAGACAAAACCGTCTAGTACGTATCTTTGA-3′ 3′-GAACTTC TGTTTTGGCAGATCATGCATAGAAACT- 5′

Aco_ARE_OligoSequence
acoARE +15′-GAAGACAAAACCGTCTAGTACGTATCTTTGA-3′
3′-CTTC TGTTTTGGCAGATCATGCATAGAAACT-5′
acoARE +45′-CTTGAAGACAAAACCGTCTAGTACGTATCTTTGACCT-3′
3′-GAACTTC TGTTTTGGCAGATCATGCATAGAAACTGGA-5′
acoARE +55′-ACTTGAAGACAAAACCGTCTAGTACGTATCTTTG ACCTC-3′
3′-TGAACTTC TGTTTTGGCAGATCATGCATAGAAACTGGAG-5′
acoARE_pal15′-TTG AAG ACA AAA CCG TCT AGA CGG TTT TGT CTT CAA-3′
acoARE_pal25′-TCA AAG ATA CGT ACT AGT ACG TAT CTT TGA- 3′
acoARE_pal1 +one each5′-CTTG AAG ACA AAA CCG TCT AGA CGG TTT TGTCTTCAAG-3′
acoARE_pal2 +one each5′-GTCA AAG ATA CGT ACT AGT ACG TAT CTT TGAC-3′
acoARE_pal1-5' over A5′-ATTG AAG ACA AAA CCG TCT AGA CGG TTT TGT CTT CAA-3′
acoARE_pal2 5' over A5′-ATCA AAG ATA CGT ACT AGT ACG TAT CTT TGA-3′
acoARE_pal1-2 each5′- G AAG ACA AAA CCG TCT AGA CGG TTT TGT CTT C-3′
acoARE_pal2-1 each5′- CA AAG ATA CGT ACT AGT ACG TAT CTT TG- 3′
acoARE_pal2-2 each5′- A AAG ATA CGT ACT AGT ACG TAT CTT T- 3′
acoARE_pal2-1-3’G5′- CA AAG ATA CGT ACT AGT ACG TAT CTT T- 3′
acoARE_pal2-1each-5’C5′- CCA AAG ATA CGT ACT AGT ACG TAT CTT TG- 3′
acoARE_pal2-1each-3’G5′- CA AAG ATA CGT ACT AGT ACG TAT CTT TGG- 3′
acoARE_pal2 −1e+GCpair5′- GCA AAG ATA CGT ACT AGT ACG TAT CTT TGC- 3′
acoARE_Pal2-3each5′-AAG ATA CGT ACT AGT ACG TAT CTT- 3′
acoARE_Pal2-1-5'CG5′-CGAAG ATA CGT ACT AGT ACG TAT CTT CG- 3′
acoARE_Pal2-1-5'GC5′- GCAAG ATA CGT ACT AGT ACG TAT CTT GC- 3′
acoARE_Pal2-1-5'TA5′- TAAAG ATA CGT ACT AGT ACG TAT CTT TA- 3′
acoARE_Pal2-1-5'AT5′-ATAAG ATA CGT ACT AGT ACG TAT CTT AT- 3′
acoARE_Pal2-1-5'GC-Mid G5′- GCAAG ATA CGT ACTG AGT ACG TAT CTT GC- 3′
acoARE_Pal2-1-5'GC-Mid GC5′- GCAAG ATA CGT ACTGC AGT ACG TAT CTT GC- 3′
Appendix 1—table 2
List of identified Streptomyces strains with homology to aco, avar1, and cyp genes involved in avenolide biosynthesis in S. avermitilis.

Strains are from the genus Streptomyces unless otherwise noted.

ReceptorAcoCyp450Strain
ADK59_29015ADK59_RS28945ADK59_RS28935XY332
ADK54_RS22575ADK54_RS22580ADK54_RS22570WM6378
SPRI_RS01555SPRI_RS01560SPRI_RS01550S. pristinaespiralis
SPRI_RS34865/spbRSPRI_RS34860SPRI_RS34870S. pristinaespiralis
AVL59_26260AVL59_RS26265AVL59_RS26255S. griseochromogenes strain ATCC 14511
ASE41_15570/scaRASE41_RS08690ASE41_RS08700Streptomyces sp. Root264
B446_03460B446_RS03395B446_RS03385S. collinus (strain DSM 40733/Tu 365)
SAZU_2710AOQ53_RS12925AOQ53_RS12915S. azureus strain ATCC 14921
tylPorf18orf16S. fradiae
TU94_00975TU94_RS00980TU94_RS00965S. cyaneogriseus subsp. noncyanogenus
AT728_21175AT728_RS06415AT728_RS06425S. silvensis
SSFG_07848SSFG_07849SSFG_07847S. viridosporus ATCC 14672
AQJ91_00095AQJ91_RS00090AQJ91_RS00100RV15
SGLAU_25540SGLAU_RS25200SGLAU_RS25210S. glaucescens
AQI88_17505AQI88_RS17500AQI88_RS17510S. cellostaticus
avaR1aco/SM007_06205cyp17S. avermitilis
BEN35_RS25960BEN35_RS25965BEN35_RS25955S. fradiae strain Olg4R
SGM_6044SGM_6045SGM_6043S. griseoaurantiacus M045
AQJ66_RS29075AQJ66_29065AQJ66_RS29080S. bungoensis
BIV23_RS09990BIV23_RS09995BIV23_RS09985MUSC 1
OP17_RS26145OP17_RS26140OP17_RS26150S. aureofaciens strain NRRL B-1286
AOK23_RS06340AOK23_RS06335AOK23_RS06345S. torulosus strain NRRL B-3889
AOK12_RS18690AOK12_RS18695AOK12_RS18685S. kanamyceticus strain NRRL B-2535
AOK14_RS28840AOK14_RS28845AOK14_RS28835S. neyagawaensis strain NRRL B-3092
JHAT_RS31450JHAT_RS31455JHAT_RS31445JHA26
IG92_RS0101750IG92_RS0101755IG92_RS0101745S. cacaoi subsp. cacaoi NRRL S-1868
IH57_RS0113175IH57_RS0113170IH57_RS0113180NRRL F-5053
TR46_RS36115TR46_RS36110TR46_RS36120Streptacidiphilus carbonis strain NBRC 100919
AWZ10_RS30605AWZ10_RS30600AWZ10_RS30610S. europaeiscabiei strain 96–14
AMK31_RS05975AMK31_RS05980AMK31_RS05970TSRI0107
OQI_RS18015OQI_RS18020OQI_RS18010S. pharetrae CZA14
AOK15_RS33540AOK15_RS33545AOK15_RS33535S. ossamyceticus strain NRRL B-3822
AOK17_RS00790AOK17_RS00795AOK17_RS00785S. phaeochromogenes strain NRRL B-1248
B079_RS0125750B079_RS0125745B079_RS0125755LaPpAH-108
AMK33_RS39295/AMK33_38290AMK33_RS39290AMK33_RS39300CB02400
ASC56_RS07050ASC56_RS07055ASC56_RS07045TP-A0356
BEK98_43205BEK98_43200BEK98_RS44190S. diastatochromogenes
SAMN04487983_101174SAMN04487983_101173SAMN04487983_101175yr375
B5181_21375B5181_21380B5181_213704F
B9W62_10200B9W62_10205B9W62_10195CS113
SAMN05216260_11022SAMN05216260_11023SAMN05216260_11021S. jietaisiensis
BN2145_RS03090BN2145_RS03095BN2145_RS03085S. leeuwenhoekii
KY5_6076KY5_6075KY5_6077S. formicae
CW362_40715/CW362_RS40740CW362_40710CW362_40720S. populi
SAMN05421806_12721SAMN05421806_12722SAMN05421806_12720S. indicus
CTU88_08915CTU88_08920CTU88_08910JV178
BX282_0700BX282_0701BX282_06991121.2
SAMN06272765_6800SAMN06272765_6799SAMN06272765_6801Ag109_G2-15
CJD44_11095CJD44_11100CJD44_11090alain-838
C3488_RS02995C3488_RS03000C3488_RS02990Ru72
C6Y14_RS06395C6Y14_06390C6Y14_RS06400A217
IF73_RS0131080IF73_RS0131075IF73_RS0131085NRRL F-5727
C6N75_16870/C6N75_RS16880C6N75_16875C6N75_RS16865ST5x
VO63_07870VO63_07865VO63_07875S. showdoensis
IF54_RS0133395IF54_RS0133390IF54_RS0133400NRRL B-3229
EW58_RS46355EW58_RS46360EW58_RS46350S. mirabilis
BG482_RS07255BG482_RS07260BG482_RS07250LUP30
STEPF7_RS00065STEPF7_RS00060STEPF7_RS00070F-7
C6376_26350C6376_26345C6376_26355P3
BS75_RS38740BS75_RS38735BS75_RS38745Streptacidiphilus albus JL83
SMA5143A_3910SMA5143A_3909SMA5143A_3911MA5143a
SLUN_38640SLUN_38645SLUN_38635S. lunaelactis
CLW08_6960/CLW08_RS34500CLW08_6959CLW08_696169
CLW15_0573CLW15_0574CLW15_057273
DC095_032510DC095_032505DC095_032515S. xinghaiensis
C8R36_7975C8R36_7974C8R36_79763212.5
CLW07_7979CLW07_7978CLW07_798067
BX279_8804BX279_8803BX279_8805Ag82_O1-9
C8R37_8029C8R37_8028C8R37_80303212.4
DT_019_27550DT_019_27545DT_019_27555SDr-06
EDD87_5077EDD87_5076EDD87_5078S. ossamyceticus
C4J65_35580C4J65_35585C4J65_35575CB09001
DI272_14555DI272_14560DI272_14550Act143
DKG34_25265DKG34_25260DKG34_25270NWU49
CLW14_9027CLW14_9026CLW14_902875
EDE03_1306EDE03_1307EDE03_1305Ag82_G5-5
E2B92_31970E2B92_31965E2B92_31975WAC05374
FE633_RS10505FE633_RS10500FE633_RS10510NEAU-C151
FGD71_RS00515FGD71_RS00510FGD71_RS00520NEAU-SSA 1
FNX44_RS06285FNX44_RS06290FNX44_RS06280OF1
E4K73_RS21075E4K73_RS21070E4K73_RS21080IB201691-2A2
EV585_RS00830EV585_RS00825EV585_RS00835BK335
EV288_RS22310EV288_RS22315EV288_RS22305BK215
DN402_06475DN402_06480DN402_06470SW4
F3T56_RS11975F3T56_RS11980F3T56_RS11970TRM68348
EV298_RS42585EV298_RS42590EV298_RS42580BK042
ESG85_RS18290ESG85_RS18295ESG85_RS18285TRM44457
EV588_RS28855EV588_RS28860EV588_RS28850BK141
B9W62_10200B9W62_10205B9W62_10195CS113
FB157_RS34410FB157_RS34405FB157_RS34415BK340
TNCT1_RS20710TNCT1_RS20705TNCT1_RS207151–11
Sri02f_RS33870Sri02f_RS33865Sri02f_RS33875S. rishiriensis strain NBRC 13407
Appendix 1—table 3
Crystallographic refinement parameters.
SeMet AvaR1AvaR1-avenolideAvaR1-DNA
Data collection
Space groupP21P21P42
Cell: a, b, c (Å)/β (o)42.0, 78.9, 130.2/93.344.4, 232.5, 87.7/92.7130.5, 130.5, 180.6
Resolution (Å)*50–2.4 (2.5–2.40)116–2.0 (2.0–1.99)130–3.08 (3.13–3.08)
Total reflections166,803570,056836,259
Completeness (%)99.9 (98.9)95.9 (65.4)100 (100)
Rsym (%)8.8 (62.8)13.4 (62.1)7.7 (127.5)
Redundancy5.2 (5.2)4.8 (5.5)15.1 (15.2)
I /σ(I)7.6 (1.8)10.5 (2.5)25.9 (2.2)
Refinement
Resolution (Å)39.3–2.425.0–2.025.0–3.09
Number reflections31,643110,86052,651
Rwork/Rfree†22.3/27.519.9/24.119.5/26.3
Number of atoms
Protein684313,31313,169
Water2491299–
DNA/ligand–1361148
B-factors
Protein54.322.9105.3
Water44.631.7–
DNA/ligand–15.882.3
R.M.S. deviations
Bond lengths (Å)0.0120.0090.010
Bond Angles (o)1.581.481.69

  1. *Highest resolution shell is shown in parenthesis.

    †R-factor = Σ(|Fobs|-k|Fcalc|)/Σ |Fobs| and R-free is the R value for a test set of reflections consisting of a random 5% of the diffraction data not used in refinement.

Additional files

Supplementary file 1

Key resources table.

https://cdn.elifesciences.org/articles/57824/elife-57824-supp1-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/57824/elife-57824-transrepform-v2.docx

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  1. Iti Kapoor
  2. Philip Olivares
  3. Satish K Nair
(2020)
Biochemical basis for the regulation of biosynthesis of antiparasitics by bacterial hormones
eLife 9:e57824.
https://doi.org/10.7554/eLife.57824