500 mhz instrument

Optical rotations were measured using a Jasco P-2000 polarimeter. UV spectra were measured using a Beckman Coulter DU-800 spectrophotometer. CD spectra were recorded using a Jasco J-1100 CD spectrometer. NMR spectra were collected using Bruker 600 or 800 MHz NMR instruments equipped with a cryoprobe and a Varian 500 MHz instrument. HRESIMS analysis was performed using an AB SCIEX TripleTOF 4600 mass spectrometer with Analyst TF software. Semi-preparative HPLC was carried out using an Agilent 1100 series HPLC or a Dionex UltiMate 3000 HPLC system each equipped with a micro vacuum degasser, an autosampler and a photodiode-array detector.

Optical rotations were measured using a Jasco P-2000 polarimeter. UV spectra were measured using a Beckman Coulter DU-800 spectrophotometer. ECD spectra were recorded using a Jasco J-1100 CD spectrometer, and IR spectra were recorded using a Thermo Scientific Nicolet 380 FT-IR spectrometer. NMR spectra were collected using both a Bruker 800 MHz NMR instrument equipped with a cryoprobe and a Varian 500 MHz instrument. The chemical shifts reported were referenced to the residual solvent peaks of CDCl₃ (δ_H 7.26 and δ_C 77.2). HRESIMS analysis was performed using an AB SCIEX TripleTOF 4600 mass spectrometer with Analyst TF software. LC-HRESIMS experiments for molecular networking were performed using a Thermo LTQ Orbitrap XL high-resolution ESI mass spectrometer coupled to Thermo U3000 HPLC system, equipped with a solvent reservoir, in-line degasser, binary pump and refrigerated autosampler. Low resolution LC-MS was performed using a Thermo Fisher Scientific ISQ mass spectrometer with an electrospray ionization (ESI) source. Semi-preparative HPLC was carried out using a Dionex UltiMate 3000 HPLC system equipped with a micro vacuum degasser, an autosampler, and a diode-array detector.

NMR spectra were acquired on a Varian 500 MHz instrument. The water signal was suppressed by pre-saturation. A total of 16384 scans was collected with a recycle delay of 2.0 s. The reaction mixture containing Y392F (28 mg/mL, 415 μM active centers) in 20 mM Tris (pH 8.0), 100 mM NaCl, 0.2 mM ThDP and 1 mM MgCl₂ (buffer prepared in D₂O to reduce the intensity of buffer peak in the region 7 – 8 ppm) was mixed with 1 mM pyruvate in the same buffer to form LThDP. The reaction was incubated on ice for 50 s and quenched with 12.5% TCA in 1 M DCl/D₂O. The mixture was centrifuged at 15,700 g for 20 min, and the ¹H NMR spectrum of the supernatant was recorded.
Similar reaction conditions were used to observe LThDP intermediates by NMR on the R478A, R420A and Y392F DXP synthase variants in the presence of d-GAP. To form LThDP, enzyme (250-300 μM) was incubated with 500 μM pyruvate on ice for 30 seconds. The reaction mixture was then further incubated on ice for an additional 30 sec with 2 mM d-GAP (R478A and R420A) or 5 seconds with 500 μM d-GAP (Y392F), then quenched with acid.