Structural and mechanistic studies of the pyridoxal 5’-phosphate-dependent enzyme serine palmitoyltransferase
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Date
29/11/2018Author
Mykhaylyk, Bohdan
Metadata
Abstract
Sphingolipids (SLs) are complex lipid-derived structures that are essential components
of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives
ceramides are known to be involved in multiple processes such as the formation of
lipid rafts, cell signalling and membrane trafficking.
The first step of SL biosynthesis is universal to all sphingolipid-producing organisms
from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase
(SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal-
5’-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of
structural homology and catalyse the decarboxylative Claisen-like condensation of
amino acids with thioester substrates. The SPT enzyme catalyses the formation of the
universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine
and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis,
SPT plays a big role in regulating natural and pathological processes.
A lot of research interest has been recently generated by SLs isolated from bacterial
members of the human microbiome and their roles in human health. Increasing
evidence suggests that some of these SLs possess immunoregulatory effects and can
have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal
gut-dwelling bacterium that belongs to a few human microbionts known to produce
unique iso-branched sphingolipids (isoSLs); these have been shown to influence the
human iNKT cell count. The production of SLs in B.fragilis is completely regulated
by a gene product BF2461.
In this work, BF2461 was expressed and purified; using a combination of UV-vis
spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography,
it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been
assessed with a range of different chain-length substrates, including less common 15
and 17-carbon chain length coenzyme A substrates. The enzyme can produce different
types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl-
CoA. However, at high levels of PCoA, a substrate inhibition is observed that might
point to a natural control mechanism employed by the bacterium in favour of
producing iso-branched SLs (isoSLs).
The structure of BfSPT has been elucidated in a complex with its amino acid substrate
L-serine. Search and analysis of putative SPTs from other microbiome-associated
bacteria that produce isoSLs show that they share high similarity with an average
amino acid conservation of 74%, suggesting they might be adapted to a particular type
of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be
structurally characterised, and the first one to have a direct impact on human health.
Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine,
some common inhibitors of the PLP-dependent enzymes. The structure
obtained in the presence of L-penicillamine provides the first direct structural evidence
of the inhibitory mechanism by a thiazolidine complex formation in the active site of
a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction
and inhibition mechanisms of BfSPT as well as opening new prospects into researching
this interesting target and its impact on the human microbiome.