In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

α-Pyrone rings exist in many polyketide synthase (PKS) derived natural products. We report the first in vitro reconstitution of α-pyrone ring formation by a type I PKS using chemically synthesized substrates.


Contents of Supporting
. 18 Table S1.  Complete manual rebuilding was performed with COOT 4 and refinement was performed using CCP4 REFMAC5 5 and Phenix Refine. 6 The statistics of data collection and refinement are summarized in Table S4. All molecular graphics figures were generated with the program Pymol. 7

Mutagenesis of
In vitro reconstitution of myxopyronin (or derivatives) formation. The one pot reaction mixture in 40 mM Tris-HCl buffer pH 7.5, glycerol 10%, 300 mM NaCl (100 L) containing 100 M CP-W5, 100 M CP-E6, 30 M MxnB and 1 mM substrates (6 with 7 or 6 with 8 or S1-S4 with 8) was incubated at 37 C for 2 h. Reaction mixtures were extracted with ethyl acetate and the organic layer was evaporated. The residue was dissolved in 100 l methanol and a 5 µl aliquot of the extract was analyzed by HPLC-MS.

LC-ESI-MS measurements for protein analysis. All ESI-MS-measurements for intact proteins
were performed on a Dionex Ultimate 3000 RSLC system using an Aeris Widepore XB-C8, 150 x 2.1 mm, 3.6 µm dp column (Phenomenex, USA). Separation of 1 µl sample was achieved using a linear gradient from (A) H2O + 0.05 % FA to (B) ACN + 0.05 % FA at a flow rate of 300 µl min -1 and 45 °C. The gradient was initiated by a 1.0 min isocratic step at 2 % B, followed by an increase to 60 % B at 8 min to end up with a 3 min step at 60 % B before reequilibration in initial conditions. UV spectra were recorded with a DAD in the range from 200 to 600 nm. The LC flow was split to 75 µl/min before entering the maXis 4G hr-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) using the standard Bruker ESI source. In the source region, the temperature was set to 180 °C, the capillary voltage was 4000 V, the dry-gas flow was 6.0 l/min and the nebulizer was set to 1.1 bar. Mass spectra were acquired in positive ionization mode ranging from 600 -1800 m/z at 2.5 Hz scan rate. Protein masses were deconvoluted by using the Maximum Entropy algorithm (Copyright 1991-2004 Spectrum Square Associates, Inc.). The gradient was initiated by a 1 min isocratic step at 5 % B, followed by an increase to 95 % B in 6 min to end up with a 1.5 min step at 95 % B before reequilibration with initial conditions. UV spectra were recorded by a DAD in the range from 200 to 600 nm. The LC flow was split to 75 µl/min before entering the maXis 4G hr-ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) using the standard ESI source. Mass spectra were acquired in centroid mode ranging from 50 -1000 m/z at 2 Hz scan speed.       (14) (also see Table S6).   Table S5. MS analysis of the loading of 6 or 8 onto MxnB, CP-E and CP-W, which were used in the competition assays.

HPLC-MS measurements to analyze the products of in vitro
Percentages are calculated using the deconvoluted mass peak heights and they are relative to the total amount of protein species present.

Protein
Substrate Product      Table S6), while only high-resolution mass spectrometry (HRMS) could be obtained for MxnEE (11).    Percentages are calculated using the deconvoluted mass peak heights and they are relative to the total amount of protein species present.

Protein
Substrate Product  of the reflection data chosen randomly and omitted from the start of refinement.).  Figure S14. Comparison between proposed mechanisms of A) CsyB and B) MxnB. While CsyB proceeds via a hydrolysis mechanism, no indication for such a mechanism was found for MxnB.

(JHS412) (E)-3-methylhex-2-enal
The title compound was prepared from 2 according to the following procedure: To a stirred suspension of PCC (4.28 g, 19.9 mmol) and NaOAc (200 mg, 3.98 mmol) in CH2Cl2 The title compound was prepared according to the following procedure: To a stirred solution of γ-amino butyric acid (  The title compound was prepared from 9 using 3 as aldehyde according to the general procedure D: Yield: 60 %, yellow oil. 1

S1-S4 Substrates Synthesis Procedure
The substrates were synthesized as previously described. 14 Scheme 3: 3-Chloroperoxybenzoic acid (75%, 27.8 g, 120 mmol) was added in small portions over a period of 50 min to a solution of ß-(-)-citronellene (13.6 g, 14.5 ml, 100 mmol) and sodium acetate (9.84 g, 120 mmol) in CH2Cl2 (250 mL) at -20 °C and stirring was continued at this temperature for 20 min. The suspension was warmed to 0 °C, stirred for 20 min and quenched by the addition of saturated aqueous NaHCO3 (150 mL). The layers were separated, the aqueous phase extracted with CH2Cl2 (3 × 50 mL) and the combined organic fractions were washed with aqueous NaOH (1M, 100 mL), dried over Na2SO4 and concentrated in vacuo to final volume of approx. 100 mL. The crude product 1 was used further without isolation and purification. (100 mL), the organic layer was separated and the water phase was extracted with Et2O (2 × 25 mL). Combined organic phases were washed with brine (2 × 100 mL), dried over Na2SO4 and evaporated. The crude aldehyde was used in the next step without purification. To a solution of 5 (500 mg, 2.1 mmol) in THF (5 mL) and water (1 mL) at room temperature was added LiOH (100 mg, 4 mmol). After stirring for 3 h, reaction mixture was added to a vigorously stirred mixture of 10% sodium dihydrophosphate solution (10 mL) and ethyl acetate (10 mL).
The organic phase was separated and water phase extracted with ethyl acetate (2 × 5 mL).
Combined organic extracts were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue (acid 6, 270 mg, 60%) was used further without purification.