Identification and characterisation of novel sphingosine-1-phosphate lyases in Burkholderia ssp.

Abstract

Sphingolipids (SLs) are ubiquitous elements in the membranes of eukaryotes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs act as potent signalling molecules involved in numerous cellular pathways having been implicated in cancer, inflammatory conditions and cardiovascular disease. A central SL signalling molecule is sphingosine-1- phosphate (SIP), generated by the phosphorylation of sphingosine by two kinase isoforms sphingosine kinase 1 and 2 (SPHK1, SPHK2). The irreversible breakdown of SIP is catalysed by sphingosine-1-phosphate lyase (SIPL), a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyses the retro-aldol-like cleavage of SIP to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine (PE). S1PL is a PLP Fold type I carbon-carbon bond lyase belonging to the Group II PLP-dependent decarboxylase subfamily and is the terminal enzyme in sphingolipid metabolism. In this work two putative SI PL genes (BPSS2021 and BPSS2025) were identified in Burkholderia pseudomallei K96243, an organism that has not been shown to produce sphingolipids, suggesting an auxiliary role for these enzymes in this pathogenic bacterium. Using a combination of UV-visible (UV-vis) spectroscopy, mass spectrometry (MS), X-ray crystallography and established assay methodology we validate that these genes encode enzymes with SI PL activity.

There are a number of published SI PL assays that rely on two main methodologies to monitor SI PL activity; MS analysis of a derivatised product or the use of fluorescent SIP substrate derivatives. Here we describe the development of two new enzyme-coupled assays that use UV-vis to measure the activity of SIPLs providing a convenient method for enzyme characterisation. The first coupled assay uses a human bone phosphatase, PHOSPHOl, which is highly specific for the PE product of the lyase reaction. This generates inorganic phosphate (Pi) which is detected using a malachite green based dye or enzymatically via the purine nucleoside phosphorylase (PNP) degradation of 2- amino-6-mercapto-7-methylpurine riboside (MESG). The second assay employs a recombinant form of human fatty aldehyde dehydrogenase (FALDH) which uses the 2E-HEX as a substrate. Activity is monitored via the reduction of the FALDH NAD+ cofactor to NADH and the concomitant increase in absorbance at 340 nm.

Using this assay we were able to kinetically characterise the two homologous B. pseudomallei SI PL isoforms (S1PL2021 and S1PL2025) and suggest that this new method could be applied to the studies of other bacterial and eukaryotic SIPLs.

We also report the determination of the x-ray structure of S1PL2021, with the PLP cofactor bound, at 2.0A resolution which provides an insight into residues potentially involved in catalysis. The SI PL reaction mechanism requires a general base to deprotonate the SIP 3'-OH. This step in the enzyme's mechanism was probed via the mutagenesis of potential residues involved to give insight into this key stage in catalysis whereby the PLP-S1P intermediate is deprotonated releasing the 2E-HEX product. Based on structural interrogation of the enzyme, candidate residues are proposed as the possible base in the Burkholderia isoforms of the enzyme.