Extended Data Fig. 4: Identification of stable PLP-pyruvic acid (PLP-PA) adduct in SAcBesB purified from Rhodococcus.
From: Terminal alkyne formation by a pyridoxal phosphate-dependent enzyme
a) Example of the orange crystals of SAcBesB. b) UV-Vis spectrum of a SAcBesB crystal, with an extended absorbance at ~ 460 nm, indicating the bound PLP-adduct also persists throughout crystallographic experiments; however, we note the spectra for this sample was not of high quality. c) Examples of possible PLP-adducts which could be bound in the crystals. d) HPLC trace of PLP released from SAcBesB by treatment with KOH. PLP and PLP-PA are chemical standards, and the PLP-PA standard includes residual PLP from the synthesis. e) LC-MS analysis of the PLP released from SAcBesB. Traces depict the extracted ion chromatograms at m/z 318, which correspond to the PLP-PA adduct. High-resolution mass spectrometry was also used to verify the chemical formula of this compound as C11H13NO8P ([M + H]+ observed: 318.0377, [M + H]+ expected: 318.0379). f) Refinement of PLP-PA adduct in the electron density present. Left shows the clear separation between the catalytic lysine and the electron density present. Middle panel shows PLP-PA fit to the electron density present. Right panel shows the density after refinement. 2Fo-Fc electron density is shown in blue, contoured at 1.0 σ. Fo-Fc electron density is shown in green and red, and contoured at 3.0 σ and -3.0 σ respectively. PLP-PA adduct created used Phenix eLBOW. g) Abbreviated chemical mechanism for formation of the PLP-PA adduct from 2-aminoacrylate.
