L arginine

FAA extraction was performed in quadruplicate from 5 mg of dried leaf tissue in 1 ml of water, with 2 mg.L^-1 cystine as the internal standard. After homogenization by vortexing, samples were incubated for 10 min at room temperature, centrifuged at 10,000 g for 10 min, and supernatants filtered through a 0.45 μm pore membrane filter (Teknokroma S.A, Spain). Calibration standards were prepared in water by spiking the 21 amino acids analyzed at concentrations from 0.25 to 10 mg.L^-1. Amino acid standards were purchased from Sigma–Aldrich (St. Louis, MO, USA), with exception of L-arginine, which was from Duchefa Biochemie (Haarlem, The Netherlands).

Recombinant SseK1, SseK2 and point mutant proteins were prepared as described and GT kinetics were measured using UDP-Glo^TM Glycosyltransferase Assay kit (Promega, #V6961) by manufacturer’s instruction. Enzyme reaction buffer (ERB) was prepared as 25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 4 mM MnCl₂, 1 mM DTT and reaction was eliminated by using the nucleotide detection buffer. For preparing the acceptor-substrate, L-arginine (Duchefa BIOCHEMIE, > 98.5% purity) was dissolved in ERB. Synthetic peptides of GAPDH (Genscript, > 90% purify) was purchased and dissolved in ERB. White 96-well plates (ThermoScientific) were used for luminescence assay and the plate was read by using luminometer (VictorX5, PerkinElmer). Kinetics parameters were calculated using GraphPad Prism5 ver.5.03 software. The points represent an average of two samples and error bars represent mean ± S.D. The final specific activity of the transfer reaction was corrected considering the hydrolysis reaction, which was performed using SseK1/SseK2, UDP-GlcNAc, and MnCl₂.

β-CD, 3-amino-3-deoxy-β-CD and 1-(p-toluenesulfonyl)imidazole were obtained from Tokyo Chemical Industry Co., Ltd. 10,12-Pentacosadiynoic (PCDA), 1-adamantanecarboxylic acid, ammonium chloride, and chloroform were purchased from Sigma-Aldrich Chemicals Co (St. Louis, Mo, USA). N-hydroxysuccinimide (NHS) was obtained from Fluka. N,N-Dimethylformamide (DMF) was obtained from Alfa Aesar, a Johnson Matthey Company. 1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) was purchased from Acros Organics, New Jersey, USA. Organic solvents such as hexane, ethyl acetate, acetone, and diethyl ether were of chromatographic purity, and the water used was triple distilled. L-arginine was purchased from Duchefa Biochemie, Haarlem, The Netherlands. Other amino acids containing L-lysine were obtained from Sigma-Aldrich Chemical Co (St. Louis, Mo, USA) and Tokyo Chemical Industry Co., Ltd.