S-adenosylhomocysteine as a methyl transfer catalyst in biocatalytic methylation reactions

Abstract

Supplementary Methods General information All methyltransferase-encoding pET28a expression plasmids were purchased from BioCat GmbH. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions. The gels were stained with Coomassie brilliant blue. Analytical high-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20AT HPLC with a Shimadzu SPD-M20A diode array detector. Phenomenex Luna ® 5 µm SCX 100 Å, LC Column 100 x 4.6 mm was used for cation exchange. Size exclusion chromatography was performed on AKTA Explorer 100 FPLC System with a HiLoad ® 16/600 Superdex ® 200 pg column. High-resolution mass spectra were obtained on a Bruker maXis 4G UHR-TOF Mass Spectrometer. Chiral HPLC-UV analysis was performed on Agilent 1100 system with CHIRAL-AGP column from Chrom Tech, Inc. 1 H NMR spectra were recorded on a a Bruker Avance Neo NMR spectrometer operating at 500 MHz proton frequency and chemical shifts were internally referenced to residual proton signals of solvents. Chemical shifts (δ) were reported in parts per million (ppm). Standard abbreviations indicating multiplicity were used as follows: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), and m (multiplet). Coupling constants (J) were reported in Hertz (Hz). Unless otherwise noted, all chemicals and reagents were purchased from Sigma Aldrich and used as recieved. Antibiotics were purchased from PanReac AppliChem. Ingredients for buffers were purchased from Acros Organics. L-Histidine was purchased Fluka TM. Myo-inositol was purchased from Apollo Scientific ltd. 2,7-dihydroxynaphthalene was purchased from FluoroChem Co. Deuterated solvents were purchased from Cambridge Isotope Laboratories. Protein expression E. coli BL21(DE3) pLysE cell: pET-28a plasmids containing genes were transformed using standard heat-shock protocols for chemically competent E. coli BL21(DE3) cells. E. coli cells containing the plasmid were collected from LB-AGAR plates with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml) and used to inoculate LB medium with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml) (20 ml). After incubation at 37°C overnight, 15 ml of pre-culture was used to inoculate fresh LB Medium (1L) with Chloramphenicol (35 µg/ml) & Kanamycin (50 µg/ml). 1 The cells were grown in 3L shaking flask at 37 °C (170 rpm) for about 4 h until OD600 reached 0.6~0.7 and then the culture was cool to 18 °C and Isopropyl β-D-thiogalactoside (IPTG) was added to final concentration of 100 µM. Expression was performed for 20 hours. Cells were harvested by centrifugation at 10,000 x g for 30 minutes and stored at-20 °C.

S‐Adenosyl‐l‐Methionine Salvage Impacts Psilocybin Formation in “Magic” Mushrooms

“…Interestingly, a simple two‐enzyme reaction with AdoK and PsiM elevated the norbaeocystin levels 2.5‐fold. We correlated this to a spontaneous hydrolysis of released SAH, which may be enhanced by downstream AdoK activity. In sum, this report provides evidence that 1) the genes of the SAM salvage cycle are co‐expressed with the psilocybin biosynthetic gene cluster and 2) SahH and AdoK can support the final step of psilocybin biosynthesis in vitro.…”

“…Adenosine kinases catalyze the phosphorylation of adenosine (Ado) to adenosyl‐5′‐monophosphat (AMP) using ATP as phosphate donor and they play an intrinsic role in purine salvage metabolism, primarily to regenerate Ado to AMP, eventually to provide new ATP in a three‐step reaction (Scheme ) . Most importantly, they are indirectly involved in the SAM regeneration system as well …”

Biocatalytic Alkylation Cascades: Recent Advances and Future Opportunities for Late‐Stage Functionalization

“…In order to circumvent this issue, a streamlined cofactor regeneration system was recently developed by Liao and Seebeck ( Figure 3C). [35] The innovative step in this work arises from converting SAH directly to SAM using HMT and methyl iodide as the Me source ( Figure 3C). This elegant two-enzyme strategy was compatible with N-, O-and C-MTases and was applied in reactions such as the C-methylation of 12 to 13.…”

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

“…We envisage that blending directed evolution strategies with cofactor analogue mapping, and new strategies to recycle the SAH product formed by Calkylation [15,44] will provide new opportunities to identify enzyme variants with wider substrate promiscuity. Key to the success of this approach is the compatibility of SalL and NovO to couple in situ generation of SAM/SAE cofactor analogues (SalL) with C-(m)ethyl transfer (NovO).…”

“…Key to the success of this approach is the compatibility of SalL and NovO to couple in situ generation of SAM/SAE cofactor analogues (SalL) with C-(m)ethyl transfer (NovO). We envisage that blending directed evolution strategies with cofactor analogue mapping, and new strategies to recycle the SAH product formed by Calkylation [15,44] will provide new opportunities to identify enzyme variants with wider substrate promiscuity. PhD studentship.A .C.w as funded by the BBSRC (BB/ P005578/1).…”