33 research outputs found
Structural Studies of an A2-Type Modular Polyketide Synthase Ketoreductase Reveal Features Controlling α‑Substituent Stereochemistry
Modular polyketide synthase ketoreductases
often set two stereocenters
when reducing intermediates in the biosynthesis of a complex polyketide.
Here we report the 2.55-Å resolution structure of an A2-type
ketoreductase from the 11th module of the amphotericin polyketide
synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final
catalytically competent ketoreductase type to be structurally elucidated.
Through structure-guided mutagenesis a double mutant of an A1-type
ketoreductase was generated that functions as an A2-type ketoreductase
on a diketide substrate analogue, setting an α-alkyl substituent
in an l-orientation rather than in the d-orientation
set by the unmutated ketoreductase. When the activity of the double
mutant was examined in the context of an engineered triketide lactone
synthase, the anticipated triketide lactone was not produced even
though the ketoreductase-containing module still reduced the diketide
substrate analogue as expected. These findings suggest that re-engineered
ketoreductases may be catalytically outcompeted within engineered
polyketide synthase assembly lines
Scheme model summarizing the ATP-dependent and phosphorylation-dependent regulation of AfsR.
Unmodified full-length AfsR is autoinhibited by its C-terminal domains. AfsR is activated by ATP binding and significantly stimulates the production of transcripts. The phosphorylation of AfsR results in increased ATPase activity, potentially counteracting the effects of ATP binding. Phosphorylated AfsR forms more oligomers and shows slightly higher transcriptional activation compared to the unmodified form. (TIF)</p
The TIC of the SARP, RNAP, and <i>afsS</i> promoter DNA.
(A) Domain structures of SARPs. The N-terminal ODB together with the following BTA is referred as an SARP domain. S. coelicolor AfsR is a large SARP with additional NOD and TPR domains. (B) Hypothetical scheme for the regulation of S. coelicolor AfsR. (C) Fluorescence polarization assays of the SARP with afs box. Error bars represent mean ± SEM of n = 3 experiments. (D) Transcription assays with increasing concentrations (62.5 nM, 125 nM, 250 nM, 500 nM, 750 nM, 1,000 nM) of the SARP. CK represents the control group without the addition of the SARP. Data are presented as mean ± SEM from 3 independent assays. (E) Assembly of the SARP-TIC. The protein compositions in the dotted line boxed fractions are shown in the SDS-PAGE. The original gel image can be found in S1 Raw Images. (F) The afsS promoter fragment used for the SARP-TIC assembly. The −35 element, −10 element, the TSS, and the 6-bp noncomplementary bubble are denoted. The afs box is colored orange and contains DRup (the upstream direct repeat) and DRdown (the downstream direct repeat). The top (non-template, NT) strand and bottom (template, T) strand are colored light green and dark green, respectively. (G) Two views of cryo-EM map. The map was generated by merging the consensus map of the full SARP-TIC and the focused map of the SARP region in Chimera X. (H) Cartoon representation of the SARP-TIC structure. The subunits are colored as in the color scheme. The data underlying C, D, and E are provided in S1 Data. BTA, bacterial transcriptional activation; cryo-EM, cryo-electron microscopy; NOD, nucleotide-binding oligomerization domain; ODB, OmpR-type DNA-binding; RNAP, RNA polymerase; SARP, Streptomyces antibiotic regulatory protein; TIC, transcription initiation complex; TPR, tetratricopeptide repeat; TSS, transcription start site.</p
Phosphorylation modulates AfsR transcriptional activation.
(A) LC-MS/MS analysis showing that S22, T337, S391, T506, and S953 are phosphorylated in AfsR by AfsKΔC. The lowercase letter in the peptide sequence indicates phosphorylated residue. “b” and “y” denote peptide fragment ions retaining charges at the N and C terminus, respectively. The subscript numbers indicate their positions in the identified peptide. (B) Sequence alignment of Streptomyces AfsR family members highlighting the consensuses sequences neighboring phosphorylation sites S22, T337, S391, T506, and S953. Orange boxes represent phosphorylation sites. (C) Transcription assays with increasing concentrations of phosphorylated AfsR by AfsKΔC (phos-AfsR) and untreated AfsR. (D) Transcription assays of 500 nM AfsR mutants mimicking dephosphorylation (T337A) and phosphorylation (S22E, T337E, T506E, S953E, and S391E/T337E/T506E/S953E (4E)). Data are presented as mean ± SEM from 3 independent assays. n.s. means no significance; *P P T337A and AfsR4E. (F) Fluorescence polarization assay of AfsR4E and dephosphorylated AfsR (dephos-AfsR) with afs box. The concentration of afs box was 10 nM. Error bars represent mean ± SEM of n = 3 experiments. The data underlying A, C, D, E, and F are provided in S1 Data. (TIF)</p
Purification and assembly of protein complexes.
(A) Assembly of S. coelicolor RNAP-σHrdB. The peak of RNAP-σHrdB was analyzed by SDS-PAGE. (B) Purification of AfsR. (C) Purification of ΔTPR. (D) Purification of SARP. (E) Purification of RbpA. (F) Purification of CarD. (G) Purification of AfsKΔC. (H) Assembly of AfsRT337A-TIC in the presence of 1 mM ATPγS. The protein compositions in the dotted line boxed fractions are shown in the SDS-PAGE. The original gel images can be found in S1 Raw Images. (TIF)</p
The original gel images contained in the figures.
Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.</div
SARP interacts with σ<sup>HrdB</sup> R4, β FTH, β′ ZBD.
(A) The SARP protomers interact with σHrdB, β, and β′. (B) The upstream SARP protomer contacts σHrdB R4 by its ODB domain. Salt bridges are shown as red dashed lines. (C) The H499 of σHrdBR4 is enfolded in an amphiphilic pocket of the BTA domain. The K496 of σHrdBR4 makes a salt bridge with the E246 of the BTA domain. (D) The downstream SARP protomer make extensive interactions with the β FTH, the preceding loop (TPL) and the following loop (TFL) of the β flap. SARP is colored orange and β flap is colored blue. Hydrogen bonds, salt-bridges, and van der Waals interactions are shown as yellow, red, and gray dashed lines, respectively. (E) Interactions between the β′ ZBD and the ODB of downstream SARP. The positively charged R67 and R69 of β′ ZBD contact the negatively charged E76 and E77 of the HTH loop of the ODB domain. (F) Mutating interfacial residues of SARP impaired transcription activation. The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3; *P P Streptomyces strains, highlighting the residues interacting with β (blue), σ R4 (cyan), and αCTD (purple). The black boxes highlight the positions conserved. BTA, bacterial transcriptional activation; FTH, flap tip helix; ODB, OmpR-type DNA-binding; SARP, Streptomyces antibiotic regulatory protein; ZBD, zinc-binding domain.</p
ATPγS activates phosphorylated AfsR and AfsR<sub>4E</sub>.
Transcription assays involving 500 nM phosphorylated AfsR (phos-AfsR) or AfsR4E, with and without preincubation with 1 mM ATP or ATPγS. CK represents the control group without the addition of AfsR. The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3. (TIF)</p
RbpA and CarD do not influence the transcriptional activation capability of SARP.
In vitro transcription assays with or without RbpA and CarD on the afsS promoter (left) and transcription assays with or without 500 nM SARP in the presence of RbpA and CarD (right). The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3. (TIF)</p
Comparison of SARP with EmbR (PDB ID: 2FEZ) comprising an additional C-terminal FHA domain.
Comparison of SARP with EmbR (PDB ID: 2FEZ) comprising an additional C-terminal FHA domain.</p