L serine

Unless otherwise specified, all reagents and materials used were obtained without additional purification from commercial suppliers. Betulinic acid (BA) (≥98%), 1-octanol, and acetonitrile (for HPLC, gradient grade) were obtained from Sigma-Aldrich (Steinheim am Albuch, Germany). L-aspartic acid, L-isoleucine, L-leucine, L-methionine, L-proline, L-threonine, and L-valine of purity ≥98% were purchased from FluoroChem (Derbyshire, UK). L-serine (≥98.5%), L-phenylalanine (≥98.5%), L-proline (≥98.5%), and L-cysteine (≥98%) were provided by Carl Roth (Karlsruhe, Germany). L-alanine (>99.0%) was acquired from Bachem (Bubendorf, Switzerland) and L-lysine (≥98.0%, anhydrous) from Glentham Life Sciences (Corsham, UK). L-phenylalanine (99%) was provided by Alfa Aesar (Ward Hill, MA, USA). L-tryptophan (>98.5%) was purchased from TCI. Thionyl chloride (99.5%) was provided by Across Organic Geel (Geel, Belgium). Ammonium hydroxide solution 25% (NH₃.H₂O) of analytical grade was purchased from StanLab (Lublin, Poland). Ethanol (99.8%) was provided by Merck (Darmstadt, Germany). Methylene chloride and DMSO of high purity were provided by Chempur (Gliwice, Poland). Deuterated dimethyl sulfoxide (DMSO-d₆) (99.8%) was obtained from Deutero GMBH (Kastellaun, Germany).

17β-estradiol, S-adenosyl-l-methionine (SAM), S-adenosyl-l-homocysteine (SAH), N⁵-methyltetrahydrofolate, tetrahydrofolate (THF), methylviologen, benzylviologen, methylcobalamin (MeCo), nicotinamidadenindinucleotide-phosphate (NADPH), l-homocysteine, l-serine, ATP (ATP), l-methionine, and methyl-¹³C-l-methionine were purchased from Carl Roth (Karlsruhe, Germany), Carbosynth (Compton Berkshire, UK), or Merck (Darmstadt, Germany). Other chemicals or reagents were of analytical or high-performance-liquid chromatographic (HPLC) grade. Denitratisoma oestradiolicum (DSM 16959) was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany). Escherichia Coli strain BL21(DE3) was purchased from New England BioLabs (Frankfurt am Main, Germany).

Hydrolysis of carbohydrates was assessed in vitro by measuring the accumulation of reducing sugar ends using the BCA assay^{42 (link)} and D-glucose as a standard for calibration (Supplementary Fig. 11). Purified enzyme was diluted to 1 mM in 20 mM (N-2-hydroxyethylpiperazine-N′−2-ethanesulfonic acid) HEPES (pH 7.5; ionic strength, 160 mM [adjusted with NaCl]) containing 10 mM MgCl₂ and 50 mM c-di-GMP (BioLog). Samples were incubated at rt for 5 min and reactions were initiated by the addition of 0.05% of the relevant carbohydrate provided by Sigma-Aldrich: glycogen from rabbit (cat. no G8876); amylose from potato (cat. no A0512); amylopectin from maize (cat. no. 10120); and pullulan (P4516). The mixture was incubated at 37 °C in a heat block for 1 h. Next, an equal volume of BCA reagent solution (250 mM Na₂CO₃; 144 mM NaHCO₃; 2.5 mM sodium bicinconinic acid (Sigma); 6 mM L-serine (Roth); 2.5 mM CuSO₄ x 5 H₂O (Roth)) was added, reactions were vortexed vigorously and incubated at 80 °C in a heat block for 30 min. Assay mixtures were then cooled to rt. 125 μl sample aliquots were transferred in duplicates to a 96 well plate and the absorbance at 562 nm was measured using a plate reader.