Exploring the structure and function of key enzymes involved in microbial sphingolipid biosynthesis
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Date
31/07/2021Author
Tang, Peijun
Tang, Gary
Metadata
Abstract
Sphingolipids (SLs) are a diverse class of lipid molecules derived from the amino
acid L-serine and long chain fatty acids (e.g. carbon chain lengths C14-C26). When
combined together these building blocks form a so-called “sphingoid”, also known as
a long-chain base (LCB). Decades of research into these enigmatic molecules has
revealed that SLs are essential components of eukaryotic cell membranes and control
many critical cellular functions. The SL biosynthetic pathway begins in all organisms
with the Claisen-like, decarboxylative condensation of L-Ser and long-chain fatty acid
acyl-CoA thioester (CoASH) substrates (most commonly C16/palmitoyl) to form the
intermediate 3-ketodihydrosphingosine (3-KDS). This first key irreversible reaction is
catalysed by the pyridoxal 5’-phosphate (PLP)-dependent serine palmitoyltransferase
(SPT). The 3-KDS product is then reduced by a NAD(P)H-dependent enzyme (KDS
reductase, KDSR) to generate dihydrosphingosine (DHS). Acylation of this molecule
with a long-chain fatty acid by a ceramide synthase (CerS) leads to the formation of
ceramide (Cer). Downstream, phosphorylation with a phosphate group can occur on
the serine-derived head group and lead to the formation of sphingosine-1-phosphate
(S-1-P). The balance between Cer and S-1-P concentrations has been proposed to
control the cell survival rate in the host system. Alternatively, sugars such as glucose
can be added to give the family of glycosphingolipids (GSLs).
In contrast to higher order species, research into SL biosynthesis in bacteria has
lagged much further behind. However recent studies have revealed a number of
important microbes that produce a range of SLs and Cers. Interestingly, SLs from the
human microbiota including those from Bacteroides fragilis, a Gram-negative
commensal bacterium from the human gut, and from the oral pathogen
Porphyromonas gingivalis have recently been shown to play an essential role in
host/microbe communication. Of note is that these bacterial SLs and Cers display
many similar structural features to their mammalian homologues. However, there
appears to be “chemical signatures” distinct to those of bacterial origin. For example,
B. fragilis SLs have iso-Me branched acyl chains which are also similar to those found
in the round-worm Caenorhabditis elegans. This suggests a PLP-dependent
branched-chain amino acid transferase (BCAT/IlvE) transfers iso-Me chains from
amino acid precursors such as L-Leu to branched-chain keto acids. To fully
understand the role of bacterial SLs and their metabolism detailed investigations of
the enzymes, pathways and metabolism are required.
In this thesis, studies of different types of the three key enzymes (SPT, IlvE/BCAT
and KDSR) involved in the core microbial SL biosynthetic pathway have been
presented. Firstly, both recombinant B. fragilis SPT (BfSPT, encoded by gene BF2461)
and P. gingivalis (PgSPT, gene PG1780) were expressed in E. coli, purified and
studied with protein UV-vis spectrometry, enzyme kinetics, inhibition assays, mass
spectrometry and protein crystallization screening. 3-KDS products were detected
derived from a range of straight-chain CoA substrates (C14-C18) and amino acids
(Gly, L-Ala and L-Ser) produced by BfSPT and PgSPT. Mutagenesis of a conserved
loop (PAVAP) in SPT homologues was found to be associated with the catalytic
efficiency of PgSPT. Also the presence of a Val353 residue in BfSPT was shown to
be essential to allow the enzyme to interact with the C16-CoA substrate. Moreover,
data suggested that the position (N- or C- terminus) of the 6His-affinity tag used to
purify SPT influenced substrate inhibition by C16-CoA. A hypothetical 3D structural
model of the PgSPT PLP:L-Ser external aldimine complex was built in order to explore
the active site and residues involved in substrate binding and catalysis. In
collaboration with Prof. Mary-Ellen Davey (Florida), the role on SL biosynthesis in P.
gingivalis was also explored and found that SLs impact on the way this pathogen
interacts with human cells.
Secondly, a branched acid transaminase P. gingivalis IlvE (PgIlvE, gene PG1290)
was expressed, purified and studied using UV-vis spectrometry to investigate
substrate binding and enzyme activity. A multi-enzyme coupled assay for PgIlvE was
developed in both the ‘forward’ and ‘reverse’ direction, studied with inhibitors such as
L- and D- cycloserine (LCS/DCS), as well as x-ray crystallography. In collaboration
with Dr. Jon Marles-Wright (University of Newcastle) the crystal structures of four
different forms of PgIlvE; the PLP-bound, internal aldimine form, LCS ring-opened
ring-closed form and the PMP form, were obtained. Two residues (F56 and Y188)
were identified which played a role in substrate binding and activity.
In the final chapter, recombinant KDSR from the yeast Saccharomyces
cerevisiae (ScKDSR) was isolated from E. coli. Kinetic parameters for a soluble,
truncated form of the enzyme were determined using the substrates KDS and NADPH.
The product C18 DHS derived from C18 KDS was detected with MALDI-ToF-MS.
Recent studies of human KDSR revealed that patients with point mutations in this
enzyme suffered from skin disorders (erythrokeratoderma). To investigate the impact
of these mutations, a series of ScKDSR mutant mimics (G176S, Y180F and G263E)
were prepared. To date there has been no crystal structure of a KDSR determined but
it is a member of the short-chain dehydrogenases/reductases (SDR) superfamily. A
homology model of the 3D structure of ScKDSR with three possible NADPH docking
positions were constructed, and residues involved in substrate binding and catalysis
were suggested.
The results in this thesis shed light on the key enzymes involved in the core
biosynthetic pathway of microbial SLs and lay down a blueprint for future studies.