|dc.contributor.author||Wadsworth, John Michael||
|dc.description.abstract||Sphingolipids and ceramides are essential components of cellular membranes and
important signalling molecules. Because of a growing appreciation for their diverse
biological roles, understanding of the biosynthesis and regulation of sphingolipids
has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT)
is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation
between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA).
This first step in sphingolipid biosynthesis is conserved in all organisms studied to
date, from microbes to man.
The fungal natural product myriocin is a potent inhibitor of SPT; however, the
molecular details of inhibition are not fully understood. Myriocin contains a long
alkyl chain and a polar head group thus it displays features of both SPT substrates.
Therefore, the prevailing hypothesis is that inhibition of SPT occurs because
myriocin acts as a mimic of a key transition state of the catalytic mechanism.
Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray
crystallography and enzyme inhibition assays it has been possible to study the
interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin
initially forms an inhibitory PLP:myriocin aldimine complex in the active site that
displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected,
slow enzymatic degradation. The mechanism for myriocin breakdown has been
elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond
producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the
active site lysine265, forming a second (suicide) inhibitory complex and rendering
the enzyme catalytically inactive.
Substitution of the active site lysine produced SPT K265A, an inactive enzyme that
did not catalyse the breakdown of the PLP:myriocin complex. However, the
determination of the crystal structure of the SPT K265A:PLP-myriocin complex
revealed that the myriocin had undergone decarboxylation. Nevertheless, this
SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism
of inhibition for the first time. The novel mechanism of myriocin degradation has
implications on the structure activity relationship (SAR) and design of drugs targeted
towards SPT, the role of feedback regulation by long chain aldehydes and further
expands the range of reactions catalysed by this important enzyme.
As well as inhibition studies the structure of bacterial SPT was also examined by
preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by
21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the
N-terminus may play a role in catalysis. Additional work has been undertaken to
study an unusual membrane-bound viral SPT, composed of two naturally fused open
reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and
the SPT1 domain at the C-terminus. To study soluble mimics of this interesting
fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant
construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding
|dc.contributor.sponsor||Engineering and Physical Sciences Research Council (EPSRC)||en
|dc.publisher||The University of Edinburgh||en
|dc.relation.hasversion||Beattie, A. E.; Clarke, D. J.; Wadsworth, J. M.; Lowther, J.; Sin, H.-L.; Campopiano, D. J., Reconstitution of the pyridoxal 5'-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis. Chem. Commun. 2013, 49 (63), 7058-7060.||en
|dc.rights||Attribution-NonCommercial-ShareAlike 4.0 International||*
|dc.title||Studies on the structure, mechanism and inhibition of serine palmitoyltransferase||en
|dc.type||Thesis or Dissertation||en
|dc.type.qualificationname||PhD Doctor of Philosophy||en