Substrate selectivity of an isolated enoyl reductase catalytic domain from an iterative highly reducing fungal polyketide synthase reveals key components of programming

The complete stereochemical course and substrate selectivity of the enoyl reductase domain from the fungal polyketide synthase squalestatin tetraketide synthase (SQTKS) have been determined.


N-Acetyl cysteamine 17
KOH (2.8 g, 49.4 mmol) was added to a solution of N, S diacetyl cysteamine (2.5 g, 15.4 mmol) in water (10 ml) at 0 °C. The reaction was warmed to RT and stirred for 50 min. The reaction was then cooled to 0 °C and the pH was adjusted to 5 with aqueous HCl (2M). NaCl was added until saturation and extracted with CH 2 Cl 2 (10 × 25 ml

Preparation of angeloyl pantetheine dimethyl ketal 8Pa
Angelic acid (8, 0.11 g, 1.00 mmol) and pantetheine dimethyl ketal (0.32 g, 1.00 mmol) were dissolved in CH 2 Cl 2 (8 ml). Then the mixture was cooled to 0 °C. Then N,N-dimethylaminopyridine (0.1 g, 0.80 mmol) and N-(3-Diethylaminopropyl)-N-ethylcarbodiimide (0.38 g, 2.00 mmol) were added to the mixture. The mixture was warmed to 25 °C and then stirred for 4 h. After that the mixture was quenched with 2M HCl (10 ml) and extracted with CH 2 Cl 2 (3 × 20 ml). The organic layer was washed with saturated NaHCO 3 (25 ml) and brine (25 ml). The product was dried over MgSO 4 and concentrated in vacuo. The crude product was purified by column chromatography (ethyl acetate). The obtained product was a yellow oil (

E-2-Ethylbut-2-enoyl pantetheine dimethyl acetal 10Pa
The reaction was carried out with 0.58 g (5.09 mmol) of 10, 1.62 g (5.09 mmol) of pantetheine dimethyl ketal, 0.97 g (5.09 mmol) of EDCI and a catalytic amount of DMAP in 10 ml CH 2 Cl 2 . The solution was dumped in 10 ml HCl (2 M) and the product was extracted with 3 × 20 ml CH 2 Cl 2 . The organic phase was washed with 1 × 50 ml of a saturated NaHCO3 solution and with 1 × 50 ml of a saturated NaCl solution. The product was a colorless oil (0.99 g, 2.38 mmol, 47 %). 1

tigloyl-pantetheine 5P
A solution of pantetheine (0.45 g, 0.80 mmol) and DTT (0.12 g, 0.80 mmol) in THF (4 ml) was stirred in a nitrogen atmosphäre at 30 °C for 3 h. After that triethylamine (0.3 ml) and DMAP (0.09 mmol, 12 mg) were added to the reaction and stirred for 15 min. Then the reaction was cooled down to 0 °C and tigloyl chloride (0.19 g, 1.60 mmol) was added to the reaction. The mixture was stirred for one hour at this temperature. The solvents were removed under a nitrogen flow. The product was purified by HPLC (acetonitrile) and concentrated under a nitrogen flow. The obtained product was a colourless oil (0.14 g, 0.40 mmol, 50 %). 1

2RS-2-methylbutyryl pantetheine 6P
A solution of pantetheine (0.15 g, 27 µmol) and DTT (0.04 g, 27 µmol) in THF (1 ml) was stirred in a nitrogen atmosphäre at 30 °C for 3 h. After that pyridine (0.1 ml) and DMAP (4 mg, 0.03 mmol) were added to the reaction and stirred for 15 min. Then the reaction was cooled down to 0 °C and (±) 2-methylbutyryl chloride (0.06 g, 54 µmol) was added to the reaction. The mixture was stirred for one hour at this temperature. The solvents were removed under a nitrogen flow. The product was purified by HPLC (acetonitrile) and concentrated under a nitrogen flow. The obtained product was a colourless oil (0.035 g, 0.10 mmol, 36 %) 1  A solution of pantetheine (0.45 g, 0.80 mmol) and DTT (0.13 g, 0.80 mmol) in THF (4 ml) was stirred in a nitrogen atmosphäre at 30 °C for 3 h. After that triethylamine (0.3 ml) and DMAP (0.01 g, 0.09 mmol) were added to the reaction and stirred for 15 min. Then the reaction was cooled down to 0 °C and crotonyl chloride (0.16 g, 1.66 mmol) was added to the reaction. The mixture was stirred for one hour at this temperature. The solvents were removed under a nitrogen flow. The product was purified by HPLC (acetonitrile) and concentrated under a nitrogen flow. The obtained product was a colourless oil (0.18 g, 0.50 mmol, 34%) 1

angeloyl-pantetheine 8P
Angeloyl pantetheine dimethyl ketal (8Pa, 0.02 g, 0.002 mmol) stirred in a mixture of acetonitrile and water (1:1) and 10 % TFA for 20 min. The reaction was followed by TLC and LCMS. After that the solvents were liphophilized. 0.03 g of the product were purified by HPLC. 1

E-2,3-Dimethylbut-2-enoyl pantetheine 9P
The acyl pantetheine dimethyl acetal (9Pa, 0.55 g ,1.32 mmol) was dissolved in CH 2 Cl 2 (2 ml). TFA (0.2 ml) was then added and the solution was stirred for 15 min at 22 °C. Thereafter, H 2 O was added and the solution was stirred for an additional 10 min at 22 °C. The solvent was then removed by blowing of N 2 flow and the product was purified by HPLC. After purification, 202.0 mg (0.54 mmol) of the colorless oily product 3c were collected. 1

E-2-Ethylbut-2-enoyl pantetheine 10P
The acyl pantetheine dimethyl acetal (10Pa, 0.99 g, 2.38 mmol) of was dissolved in CH 2 Cl 2 (2 ml). TFA (0.2 ml) was then added and the solution was stirred for 15 min at 22 °C. Thereafter, H 2 O was added and the solution was stirred for an additional 10 min at 22 °C. The solvent was then removed by blowing of N 2 flow and the product was purified by HPLC. After purification, 261.0 mg (0.70 mmol) of the colorless oily product were collected. 1

E-dec-2-enoyl pantetheine 24P
E-Dec-2-enoyl chloride was prepared from E-dec-2-enoic acid (24, 0.138 g, 1.00 mmol,) with thionyl chloride (0.110 g, 1.00 mmol) in THF (0.5 ml). This solution stirred for 24 h under nitrogen, and solvent removed in vacuo. A solution of pantetheine (0.30 g, 0.54 mmol,) and DTT (0.08 mg, 0.54 mmol) in THF (3 ml) was stirred in a nitrogenatmosphäre at 30 °C for 3 h. After that pyridine (0.3 ml) and DMAP (12 mg, 0.09 mmol,) were added to the reaction and stirred for 15 min. Then the reaction was cooled to 0 °C and E-dec-2-enoyl chloride was added to the reaction. The mixture was stirrred for one hour at this temperature. The solvents were removed under a nitrogen flow. 6 mg of the product were purified by HPLC. 1  The progress of the reaction was monitored by LCMS. After 24 h further NADPH (8 mg, 10 mmol) was added to the reaction. After a further 17 h the protein was precipitated with CH 2 Cl 2 (1 ml) and centrifuged (3000 rpm, 20 min). The organic layer was removed and the aqueous layer was further extracted with CH 2 Cl 2 (2 × 1 ml). The organic fractions were combined and the solvent was removed under a flow of dry N 2 and then dried in vacuo for a further 2 h. To the dried sample was added water (450 µl) and aqueous NaOH (2M, 50 µl). The sample was left for 90 min at RT and then acidified with aqueous HCl (2M, 100 µl) to pH 3 and extracted with CDCl 3 (4 × 200 µl). The organics were combined, dried with MgSO 4 and then filtered through MgSO 4 directly into an NMR tube. (1R,2R)-(+)-1,2 diphenylethylenediamine was titrated (100 mM stock) to optimize resolution of the obtained spectra. 29 A solution of sodium bicarbonate (6.3 mg, 75 µmol), D 7 -glucose (8.0 mg, 45 µmol) and NADP + (16.7 mg, 23 µmol) were titrated to a pH 7.5 in water (1.5 ml). Glucose dehydrogenase from Pseudomonas sp (35 U) was added and the reaction was incubated at RT and the progress of the reaction was monitored by UV (340 nm). On completion the reaction was confirmed by MS (747 (100, [M]H+ ) and was used without further purification (final concentration of NADPD 15 mM). 30 A stock solution of alcohol dehydrogenase NADP + dependent, from thermoanerobium brockii (TbADH) was prepared in tris pH 7.2, 25 mM (0.085 U/µl) and was added (25 µl) to a solution of NADP + disodium trihydrate (17. mg, 20.8 µmol), isopropanol (300 µl) and tris buffer pH 9 (25 mM, 7.5 ml), this solution was incubated at 43 °C and the progress of the reaction was monitored by UV (340 nm). When no further change in absorption was observed the acetone and remaining isopropanol was removed in vacuo and the product was confirmed by MS (747 (100, [M]H+ ). This was used without and further purification (final concentration of NADPD 3.1 mM). 31 2-Methylbutyric acid (102 mg, 1.0 mmol) and S-methyl mandelate (208 mg, 1.3 mmol) were stirred at 0 °C in CH 2 Cl 2 (3 ml). To this was added DMAP (4 mg) and EDCI (240 mg, 1.25 mmol) and this was then stirred at RT and followed by TLC. On completion, aqueous HCl (0.5 M, 5 ml) was added and this was extracted with CH 2 Cl 2 (3 × 5 ml). The organic fractions were combined and concentrated in vacuo. The resulting oil was purified by column chromatography (hexane:ethyl acetate 3:1) to yield the desired product as a inseparable mixture of diastereomer (A:  Ethyl 3R-hydroxyl 2R-methylbutyrate 32 32 BuLi (1.6 M in hexanes, 4.2 ml, 6.7 mmol), was added dropwise to a solution of freshly distilled diisopropylamine (894 µl, 6.4 mmol) in THF (6.5 ml) at -78 °C. The resulting solution was stirred for 1 hour. A solution of ethyl (R)-3hydroxybutyrate (400 mg, 3.0 mmol) and hexamethylphosphoramide (900 µl) in THF (2 ml) was added dropwise to the

SQTKS isolated ER domain
The sequence of SQTKS 14 was used to design an E. coli optimised sequence which was used as the template for the amplification of the ER domain. Primers for the ER domain with added bases in bold and restriction sites underlined. FW-Nde1: CAC CAT ATG GAA CCC TTT CAT CAG CCG GGG AAG C RV-BamH1: AAA GGA TCC TTA TGG CGC GGT GAT GAC AAT TTT GC. The PCR product was cloned into pET28a and the plasmid transformed into E. coli BL21 DE3 (Novagen), 100 µl of the culture was spread on a LB agar plate with kanamycin and this was incubated overnight at 37 °C. A single colony was taken to form a starter culture (50 ml 2TY media with kanamycin added), which was incubated overnight at 37°C with shaking. The starter culture was then used to inoculate 2TY media (1:100 starter culture:media dilution per flask). The OD was monitored and when it reached ~0.6 A the flasks were cooled to 16 °C and IPTG solution (1 M, 50 µl, 0.5 mM final IPTG concentration) and the flasks were left overnight at 16 °C with shaking (250 rpm).
On completion the media was centrifuged (7000 rpm, 15 min) and the pellet was collected. The cells were resuspended in nickel column wash buffer (50 ml buffer per 1 l of media, [50 mM Tris pH 8, 150 mM NaCl, 10% glycerol (v/v) and 20 mM imidazole]). The suspension was then sonicated (6.5 min, 30 seconds 20% power, 30 seconds rest) on ice and then centrifuged (17000 rpm, 30 min). The supernatant was taken, filtered (0.45 µm filter) and purified on a Ni 2 + affinity column using a linear gradient with the elution buffer [50 mM Tris pH 8, 150 mM NaCl, 10% glycerol (v/v) and 0.5 M imidazole] to 100%. The fractions were analyzed by SDS page and fractions containing protein of approximately the right mass were combined and concentrated to ~2 ml using centrifugal ultrafiltration (10 kDa cut-off).
The concentrated protein was purified using size exclusion chromatography. The protein was loaded directly onto the column and eluted in the size exclusion elution buffer [50 mM Tris pH 8, 150 mM NaCl, 20% glycerol (v/v)]. The fractions were analyzed by SDS page and fractions containing protein of approximately the right mass and high purity were combined together and concentrated to ~2 ml using centrifugal ultrafiltration (10 kDa cutoff). The concentration of protein was estimated using a Bradford assay and the activity (U) of the enzyme was measure. The protein was then divided into aliquots (0.2 U/µl) and stored long term at -80 °C.  The identity og the protein was confirmed by MALDI analysis of digested protein fragments obtained directly from the SDS-PAGE gel.

2.2
Defining and measuring the activity units for the isolated ER domain 1 unit of activity is defined as 1 µM⋅min -1 tigloyl pantetheine reduction by NADPH at 30 ˚C. The freshly produced protein was analyzed by a standard UV assay (section 3.2.2) and the produced enzyme was diluted to the appropriate concentration (typically 0.2 mU/µl) with the size exclusion buffer.

Bradford Assay
Standard solutions of bovine serum albumin (0.1-2 ml/ ml) in size exclusion buffer [50 mM Tris pH 8, 150 mM NaCl, 20% glycerol (v/v)] were prepared by serial dilution. 100 µl of the standards were mixed with Bradford dye reagent (1 ml) and incubated for 15 min at RT. The absorption of each sample was measured at 595 nm against a standard (size exclusion buffer 100 µl, Bradford dye reagent, 1 ml) to construct a standard concentration curve. A sample of the protein to be quantified (20 µl) was diluted in size exclusion buffer (80 µl) and treated with Bradford dye reagent (1 ml). This was incubated at room temperature for 15 min and then the absorption was measured at 595 nm. This was compared to the previously prepared concentration curve to calculate the amount of protein that had been produced.

Evidence for Dimeric State of ER in Solution
The protein solution was loaded onto the column (GE Healthcare HiLoad 26/600 Superdex 200 pg) and eluted with 50 mM Tris pH 8,150 mM NaCl,20% glycerol (v/v). In the run no concentration change take place. The flow of the FPLC was constant 1mL/min over 300 min.

Calibration:
For the calibration a mixture of three different proteins (Carbonic Anhydrase, BSA, Apoferritin) was loaded on the column. The mixture was loaded onto the column (GE Healthcare HiLoad 26/600 Superdex 200 pg) and eluted with 50 mM Tris pH 8,150 mM NaCl,20% glycerol (v/v). The flow of the FPLC was constant 1mL/min over 300 min.

LCMS assay of ER
A sample of reaction mixture (20 µl) was mixed with acetonitrile (80 µl) to precipitate the protein and then centrifuged (13,000 rpm, 1 min). The supernatant was analyzed directly by LCMS using a standard LCMS profile (Section 6.1).

ER Homology Model:
A homology of the ER domain with NADP bound was generated using the SWISS-MODEL protein structure homology-modelling server in the fully automated mode. 33 The PDB entry 2VZ9 was used as the template structure. 34 Pantetheine Docking: Ligand structures were generated using Chem3D Pro 13.0 (Perkin Elmer). The ligands were docked manually into the active site using PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schrodinger, LLC.) and the resulting protein-ligand complex was energy minimised using the YASARA energy minimization server. 35 The docked ligand was removed from the resulting protein model and re-docked using Autdock Vina 1.1.2. 36 The ligand files and ER PDB file were converted into PDBQT format using Autodock Tools 1.5.6 37 to add polar hydrogens and set rotatable bonds. Ligands were docked using a grid of 44 Å (x) x 22 Å (y) x 16 Å (z) centred on the active site at position 45.8 (x), 143 (y), 76.556 (z). Images were processed using PyMol.
pdb files of representative docked structures are appended as supplementary information.

Integration of Angelic Mandelate Spectrum
Integration of crosspeaks for 30 from reduction of 8P was achieved using MestreNova 9.0 Software using standard parameters.