PhD student profiles

     
     
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Benjamin Lichman
Wellcome Trust 4-year Interdisciplinary PhD Programme, beginning in Autumn 2011

Project title
Understanding the mechanism and revealing the biocatalytic potential of norcoclaurine synthase

Principle investigator: Professor John Ward, Department of Biochemical Engineering, UCL
Co-investigator: Professor Helen Hailes, Department of Chemistry, UCL

 
Background
I graduated from the University of Cambridge with an MSci in Natural Sciences. During my final two years I studied chemistry, mostly focussing on organic chemistry, chemical biology and biophysics. My final year masters project was on protein folding pathways in the lab of Professor Jane Clarke. Through my degree I became particularly interested in the chemical and physical basis of enzyme activities; this interdisciplinary PhD programme was an ideal opportunity to explore these interests further.
 
Rotation projects

Rotation 1
Novel alkaloid synthesis using phosphate mediated reactions. Supervised by Prof Helen Hailes.
During this chemical biology project I learnt numerous organic chemistry techniques including synthesis, flash chromatography and analytical methods such as NMR and LCMS.

Rotation 2
Understanding the mechanism and unlocking the biocatalytic potential of norcoclaurine synthase. Supervised by Prof John Ward. This biochemistry project featured norcoclaurine synthase (NCS), the enzymatic alternative to the phosphate mediation reactions I was introduced to in the first rotation project. Here I made mutants of NCS in order to investigate its mechanism and expand its substrate scope. This project became my main PhD project.

Rotation 3
The physicochemical basis of protein promiscuity. Supervised by Dr Irilenia Nobeli.
The computational project introduced me to bioinformatics but also crucially gave me widely applicable skills such as protein structure analysis, scripting and statistical programming. In this project I used publically available data on kinase structures and drug interactions to build models of protein binding promiscuity.

 
PhD Project

Norcoclaurine synthase (NCS) catalyses the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) via a Pictet-Spengler condensation (Figure 1). In plants, (S)-norcoclaurine is the precursor to all benzylisoquinoline alkaloids (BIA), a family of natural products which include many bioactive compounds; for example, morphine is a famous analgesic, berberine has anticancer properties and noscapine is an antitussive drug.

 
figure 1
Figure 1: NCS activity in plants. Dopamine and 4-HPAA are converted to (S)-norcoclaurine. Protein structure is from PDB 2VQ5.
 

My PhD project has focussed entirely on NCS, and has had three distinct parts. Firstly, I have been attempting to understand the mechanism of the enzyme, primarily using site-directed mutagenesis. Secondly, I have been producing mutants of NCS which can accept non-natural substrates; these will enable the formation of novel chiral alkaloids. Finally, I have combined NCS with other enzymes in vitro in chemo-enzymatic cascades to synthesise complex alkaloids in an efficient and green manner (figure 2).

 
figure 2
Figure 2: Schematic of two-enzyme one-substrate one-pot chemoenzymatic cascade for the synthesis of chiral alkaloids, using NCS in a key step. TAm = transaminase, CH2O = formaldehyde, ee = enantiomeric excess, h = hours.
 

This project has involved diverse techniques. These have included computational methods such as molecular docking and molecular dynamics. Typical biochemical methods used include mutagenesis, enzyme purification and kinetics. There have also been forays into organic chemistry and crystallography. The multi-disciplinary nature of this project has been enabled by the resources and spirit of collaboration in the ISMB.

 
Publications

Lichman, B.R., Gershater, M.G., Lamming, E., Pesnot, T., Sula, A., Keep, N.H., Hailes, H.C. and Ward, J. M., ‘Dopamine-first’ mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile. (2015) FEBS Journal, 282, pp. 1137-1151

Lichman, B.R., Lamming, E., Pesnot, T., Smith, J.M. Hailes, H.C. and Ward, J. M., One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine. (2015) Green Chemistry, 17, pp. 852-855

Lichman, B.R., Lamming, E., Ward, J. M. and Hailes, H.C., Norcoclaurine synthases for the biocatalytic synthesis of tetrahydroisoquinolines. Practical Methods in Biocatalysis and Biotransformations 3. In press.

*Kwa, L. G., Wensley, B.G., Alexander C. G., Browning S. G., Lichman B. R. and Clarke J. (2014). The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus, Unlike Its Homologous Neighbours. (2014) Journal of Molecular Biology, 426 (7), pp. 1600-1610.

*Diep, C.Q., Ma, D., Deo, R.C., Holm, T.M., Naylor, R.W., Arora, N., Wingert, R.A., Bollig, F., Djordjevic, G., Lichman, B., Zhu, H., Ikenaga, T., Ono, F., Englert, C., Cowan, C.A., Hukriede, N.A., Handin, R.I., Davidson, A.J. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. (2011) Nature, 470 (7332), pp. 95-101.

*these publications are not from my PhD project

 

 

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