期刊
FEBS JOURNAL
卷 282, 期 6, 页码 1137-1151出版社
WILEY
DOI: 10.1111/febs.13208
关键词
alkaloid biosynthesis; biocatalysis; enzyme engineering; enzyme kinetics; enzyme mechanism
资金
- Wellcome Trust
- EPSRC
- GlaxoSmithKline
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/G014426/1, BB/F016948/1]
- BBSRC [BB/F016948/1, BB/G014426/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [1104891, BB/G014426/1] Funding Source: researchfish
Norcoclaurine synthase (NCS) () catalyzes the Pictet-Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both invitro biocatalysis and invivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the dopamine-first' mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity.
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