Pyridoxal 5 phosphate plp

For the procedure of cloning, hetero-expression and purification of GadA and GadB from Lb. brevis 145 and GadB from Lb. plantarum WCFS1, please refer to Supplementary Materials and Methods for details. The effects of pH (3.0–6.6) and temperature (30–90°C) on the purified GADs including Lb. plantarum GadB, Lb. brevis GadB, and Lb. brevis GadA and its mutants (Lb. brevis GadA Δ5 and Lb. brevis GadA +GSHM) were carried out. Low concentrations of PLP such as 20 μM is sufficient to activate the activities of both Lb. plantarum GadB and Lb. brevis GadB (Fan et al., 2012 (link); Yu et al., 2012 (link); Shin et al., 2014 (link)), thus the reaction mixture consisted of 5 μL of 1 M monosodium glutamate (MSG; Sigma), 5 μL of 2 mM pyridoxal 5′-phosphate (PLP; Sigma), 500 μL of McIlvaine (citrate-phosphate) buffer, and 10 μg of GAD. Previous studies have indicated the PLP- and sulfate ion-dependent activation of Lb. brevis GadA and its homologous Lb. zymae GadB (Ueno et al., 1997 (link); Park et al., 2014 (link)); thus, for Lb. brevis GadA and its mutants, the reaction mixture contained 5 μL of 1 M MSG, 5 μL of 20 mM PLP, 500 μL of sulfate buffer (0.8 M sodium sulfate and 50 mM sodium acetate; different pH), and 10 μg of GAD. The kinetics of these enzymes was determined under their optimal pH and temperature, and the concentrations of substrate (glutamate) ranged from 0.9 to 36 mM.

The chemicals and reagents used in this study came from the following sources: L-methionine derived from (Merk, Germany). Sodium methanethiolate was purchased from Sigma-Aldrich, Commassi Brilliant Blue G-250, 5,5-Dithiobis-2-nitrobenzoicacid (DTNB), pyridoxal-5-phosphate (PLP), and bovine serum albumin (BSA) (Sigma, St. Louis, USA). Pharmacia Biotechnology provided Sephadex G-100 and DEAE-cellulose (Sweden). Potato dextrose agar (PDA) (Conda lab, Spain). The American Type Culture Collection provided cancer and normal cells for this study (ATCC, Rockville, MD, USA). The rest of the compounds are of the highest analytical quality.

Pyridoxal 5'-phosphate (PLP) was purchased from Sigma-Aldrich (Saint Louis, MO). The plasmid containing D-2-hydroxyisocaproate dehydrogenase (HO-Hxo-DH) gene was a kind gift of Dr. K. Muratore (University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, CA, United States).

Sodium alginate (SA), phenylboronic acid (PBA), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (Hepes) buffer (S)-α-methylbenzylamine ((S)-α-MBA), sodium pyruvate (PYR), acetophenone (ACP), L-alanine (L-ALA), and pyridoxal 5′-phosphate (PLP) were all from Sigma Aldrich (St. Louis, MO), CaCl₂ was from Carlo Erba reagents (Milan, Italy), polyvinyl alcohol (PVA, MW = 13,000–23,000 Da) was purchased from Acros Organics (Morris Plains, NJ). The enzyme amine transaminase (ATA-v1) was provided by c-LEcta GmbH (Leipzig, Germany).

Thiaminediphosphate (TDP) and pyridoxal-5’phosphate (PLP) were purchased from Sigma-Aldrich (St. Louis, USA).
Deuterium labelled internal standards TDP (thiazole-methyl-D₃) and PLP (methyl-D₃) were supplied by Cambridge Isotope Laboratories Inc. (Tewksbury, USA).
Perchloric acid (70%), hydrochloric acid and ammonium acetic acid, pro analyse were obtained from Merck (Darmstad, Germany), ammonium carbonate (>30.0% NH₃ basis) from Sigma-Aldrich and methanol (100% gradient grade) from Merck.
All reagents were of high purity grade designated for high performance liquid chromatography and mass spectrometry.
Water was obtained from a demi water system of Ovivo, Mettler-Toledo B.V. (Zurich, Switzerland).
Leftover blood samples (n = 12) were collected from patients who presented to the hospital for laboratory analyses of TDP and PLP. Blood samples were pooled and used as matrix material for the calibration curve. The initial values of TDP and PLP were 78 nmol/L and 55 nmol/L, respectively, as established by standard addition method on the UHPLC-MS/MS. Part of the pooled whole blood sample was spiked with TDP 75 and 150 nmol/L and with PLP 50 and 100 nmol/L, aliquoted and stored at -80°C until use for recovery experiments.

Ketones 1–3a, racemic amines 4–6b and pyridoxal-5′-phosphate (PLP) were purchased from Sigma-Aldrich (Steinheim, Germany). EziG³ (Fe Amber) enzyme carrier material was provided by EnginZyme AB (Solna, Sweden). Further details for equipment and analytical determination are presented in Section 4.6. All reaction solvents were degassed before use. All of the water-equilibrated solvents were prepared by shaking hydrate salt pairs in the organic solvent for 1 h at RT.