Lichman, BR; (2016) Norcoclaurine synthase: the mechanism and biocatalytic potential of a Pictet-Spenglerase. Doctoral thesis , UCL (University College London).

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Abstract

In plants, the enzyme norcoclaurine synthase (NCS) catalyses the formation of (S)- norcoclaurine via the Pictet–Spengler condensation of dopamine and 4- hydroxyphenylacetaldehyde (4-HPAA). (S)-Norcoclaurine is the precursor to all benzylisoquinoline alkaloids (BIAs), a diverse group of over 2500 natural products. The aim of this project was to elucidate the mechanism of NCS in order to enable the rational engineering of NCS activity. Variants of NCS were screened for activities with various substrates, forming novel tetrahydroisoquinolines (THIQs). NCS was combined with other enzymes in biocatalytic cascades to produce THIQs. Initially, an N-terminally truncated NCS from Thalictrum flavum (Δ19TfNCS) was expressed. Problems with the purification of Δ19TfNCS led to the use of a different truncate, Δ29TfNCS. This enzyme and variants were expressed and purified. The effect of mutations on the activity, kinetics and substrate specificity of Δ29TfNCS led to the conclusion that NCS operates with a ‘dopamine-first’ mechanism. Computational analysis, including molecular dynamics and docking experiments, supported this conclusion. Furthermore, rational engineering of substrate specificity was demonstrated. Next, the biocatalytic potential of NCS was investigated. Biotransformation conditions, such as enzyme or lysate loading, were optimised before demonstrative examples of milligram scale biotransformations were performed. Then, NCS and a transaminase were combined in a one-pot ‘triangular’ biocatalytic cascade to produce chiral BIAs. An additional chemical step led to the one-pot formation of chiral tetrahydroprotoberberines (berbines). The cascades were demonstrated on a milligram preparative scale. Methods for screening NCS mutants were examined: various enzyme preparations, reaction conditions and reporter systems were tested and evaluated. Subsequently, NCS mutants were screened for activities with numerous amines and aldehydes. NCS activity was identified with some α-substituted aldehydes and ketones. Selected mutants demonstrated an increase in activity compared to wild-type for these unusual substrates. Notably, high conversions were revealed for cyclohexanone derivatives. A number of resulting cyclohexane-spiro-THIQs were characterised.

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