Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032)

Abstract

Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

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

“…Mechanistically, NAD + first oxidizes the 3′‐OH group of SAH to facilitate efficient hydrolysis of SAH and the release of HCy. After addition of a water molecule to the remaining 3′‐keto‐4′,5′‐dihydroadenosine, the final reduction of the 3′‐keto group requires NADH+H + to give Ado . Previous works suggested the C‐terminal lysine K425 (from the adjacent subunit) to properly position the nucleoside moiety of NAD + via 2′‐OH and 3′‐OH .…”