Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae

Abstract

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

“…In contrast, surprisingly, in the multi-subunit, membrane-bound human homolog of the enzyme, no KIE is observed for the same substrate. Our results are in agreement with a recent report by Hannun and colleagues on the yeast SPT complex (34) and suggest that there are subtle mechanistic differences between the cytoplasmic and membrane-bound forms of the enzymes that are dependent on the activating small subunit.…”

“…This is due to the fact that these energy differences result in increased bond strength which can be observed as an effect on the rate of an enzyme catalysed reaction -a kinetic isotope effect (KIE). As such, this effect has been used to great effect in mechanistic studies (32)(33)(34)(35).…”

Seipin negatively regulates sphingolipid production at the ER–LD contact site

“…**, P < 0.01; ***, P < 0.001. (Ren et al, 2018). Manipulating Tsc10 level by these approaches had a profound and opposite impact on the seipin-Tsc13 interaction, without an apparent effect on the seipin-Lcb1 interaction (Fig.…”

“…The MS was operated with positive or negative ion modes, and we set the full Fourier-transfer MS scan at m/z 100-1,200, resolution 60,000. The sphingolipid species were quantified on the basis of Guan and Wenk (2006) and Ren et al (2018) with Xcalibur software (Thermo Fisher Scientific), and Microsoft Excel was used for statistical analysis.…”

Dynamics of sphingolipids and the serine-palmitoyltransferase complex in oligodendrocytes during myelination

“…SPT activity was measured in total brain membranes using a sensitive and specific mass spectrometry-based assay (26). Interestingly, activity increased from day 2 to day 5 after birth and declined thereafter ( Figure 5, Panel A).…”