Chemical structure and diversity of sphingolipids. A: Sphingolipids d18:1^Δ4trans. The module R is an amine-linked fatty acid in sphingomyelin (SM) and ceramide (CER) and an H in sphingosine (SPH) and sphingosine-1-phosphate (S1P), whereas X is either a phosphocholine in SM (see panel B), an H in CER and SPH, or a phosphate group in S1P. Phytosphingolipids carry a hydroxyl group at position 4, whereas 1-deoxy-sphingoid bases (DSBs) lack the hydroxyl at position 1. B: Sphingomyelin d18:1^Δ4trans/18:0. For details about nomenclature, see Pata et al.4 Although the singular is used in the text, each class of sphingolipid is composed of a variety of molecules differing in length, degree of unsaturation, and substitution of the fatty acid and sphingolipid chains.

Cellular locations of sphingolipids and S1P signaling modes. The color code for the various sphingolipids is the same as in Figure 2. The dashed arrows encompass reactions that are not displayed for clarity (see Fig. 2). The left part of the scheme illustrates the formation and degradation of sphingolipids in the ER, the subsequent transport of CER to the Golgi via CERT, the formation of Glyco-SPHL and SM therein, their transport to the PM via GLTP and vesicles, and their transport into the blood and their turnover via endosome. The ER- and Golgi membrane-interacting domains of CERT are represented as black hexagons, the CER-binding START is depicted in light orange, whereas the linkers between each domains are represented as black lines. In the cell, some sphingolipids also reside in the mitochondrion membrane. ABC transporters responsible for the export, import, or flipping of various sphingolipids were identified in different cellular compartments.7,8 The upper right part of the scheme depicts the extracellular transport of S1P in the blood, the stimulus-driven production of S1P (for example via binding of an antagonist to its cognate receptor), the putative intracellular transport modes of S1P, and the S1P extracellular signaling mode via the specific GPCRs S1PR_1–5.

Proposed SPL reaction mechanism.82 In the resting state, a lysine (311 in StSPL) forms a Schiff base with the aldehyde group of PLP (step I). The incoming substrate (S1P in the scheme, step II) triggers transaldimination and replaces the lysine as Schiff base partner of PLP. The resulting S1P-PLP complex is called external aldimine [step III, model presented in Figure 4(B), colored green]. After retro-aldol cleavage (step III), the long-chain aldehyde product (Hex in the scheme) leaves the active site (step IV) and a quinonoid intermediate is formed (step V). A stereospecific reprotonation (step VI) of this intermediate yields the second external aldimine (step VII). We propose that one of the two conserved lysines strictly required for activity carries out the retro-aldol cleavage in step III as well as the reprotonation (step VI). PE is then released (step VIII) and the internal aldimine is re-formed (step I).

Proposed SPT reaction mechanism.119,121 In the resting state, the PLP is linked to the enzyme via K265 (in SpSPT). Please note that the imidazole ring of H159 pi-stacks the pyridine ring of the PLP [see Fig. 6(B)]. For clarity, this residue is not shown in the subsequent steps (see comment below). The incoming amino acid (in this case serine) enters the active site (step II) and forms an external aldimine with PLP (step III). The external aldimine undergoes a nucleophilic attack, most likely by the internal aldimine-forming K265, after the second substrate, the acyl-CoA or the acyl-ACP (in this case palmitoyl), has entered the active site (step IV). The active site is expected to undergo conformational changes upon binding of the conjugated acyl. After the nucleophilic attack, a first quinonoid intermediate is formed (step V), the acyl carrier (either reduced CoA or ACP) leaves the active site, and a second external aldimine (with a β-keto acid) is formed (step VI). Decarboxylation of this intermediate produces the second quinonoid intermediate (step VII), which gets reprotonated to form the third external aldimine of the reaction cycle, between PLP and the reaction product (step VIII). The product 3-keto-dihydroSPH is then released (step IX). The proposed mechanism is based on the work of the Campopiano group.111,119,121 Importantly, H159 stabilizes by H-bond either the carboxyl group of the PLP-bound Ser (step III) or the carbonyl of the conjugated acyl (steps IV and V) and of the intermediates depicted in steps VI to VIII. For further explanations on the mechanism and especially the role of H159, please refer to the review by Ikushiro and Hayashi.106

Putative membrane-bound SPL. Proposed topology of the membrane-bound mammalian and yeast SPL homodimer.82 StSPL containing a modeled PLP-S1P external aldimine is colored as in Figure 4 and displayed as surface. A: Compared to Figure 4(A), the protein was rotated 90° anticlockwise along an axis parallel to the plane of the sheet such that the putative active site entrance faces the reader. B: The protein is oriented as in Figure 4(A). TMH stands for transmembrane helix.

Overview of the sphingolipid metabolism. The direct pathway from entry to exit, that is, from SPT to SPL, goes from the gray and purple boxes until the black and green boxed rectangles. See text for abbreviations. FA CoA, fatty acyl-Coenzyme A; DES, dihydro-SPH desaturase; PC, phosphatidylcholine; DAG, diacylglycerol; CK, CER kinase; C1PP, C1P phosphatase. See Figure 3 for the cellular location of the reactions. Dashed boxes and arrows correspond to the respective dashed arrows in Figure 3.

X-ray structure of StSPL. A: Dimeric structure of WT StSPL (PDB code 3MAD82). Subunit A is colored light gray, the subunit B dark gray, and represented in cartoon and ribbon, respectively. PLP and the covalently bound K311 as well as a phosphate ion appear in ball-and-stick in the color of the corresponding subunit and atom colors and are labeled in subunit A. Residues 1–57 are not visible and are represented by dashed lines in the color of the corresponding subunit. The N- and C-termini are indicated. The subunits are related by a twofold axis perpendicular to the plane of the sheet, indicated as a red-framed black ellipse. B: Stereo representation of the active site of subunit A showing the modeled external aldimine PLP-S1P in green (corresponding to step III in Figure 5). Residues important for activity appear in ball-and-stick in the color of the subunit. The asterisk denotes a residue belonging to the subunit B. Details about the modeling are given in Ref.82. This and the following structural figures were prepared with PyMOL.94

X-ray structure of SpSPT (PDB code 2JG2)¹¹⁷. A: Same as Figure 4(A) for holo-SpSPT. Subunit A is colored pale gray, whereas subunit B is dark green. The first 20 and the last nine residues of each subunit are represented by dashed lines. The bound Mg ions appear as brown spheres. B: Stereoview of the active site. The internal aldimine of subunit A as well as residues important for activity are shown as in Figure 4(B).

Hereditary sensory and autonomic neuropathy type 1 (HSAN I)–linked mutations on SpSPT. A: Surface representation of holo-SpSPT (PDB code 2JG2117). Subunit A is colored pale gray, whereas subunit B is dark green. The first 20 residues of subunit B are represented by a green dashed line. The residues of SpSPT corresponding to mutations linked to HSAN I are colored yellow and atom color. The bound Mg ions appear as brown spheres. Compared to Figure 6(A), the dimer was rotated 90° anticlockwise along an axis parallel to the plane of the sheet. This figure is linked to Table I. B: Stereoview of the active site. The residue G268 is colored yellow.

Putative membrane-bound SPT. Proposed topology of the membrane-bound mammalian and yeast SPT heteromer based on the assumption of Hornemann et al.140 SpSPT dimers in surface representation are colored green, brown, blue, and gray where SPTLC1 is colored with the darker shades and SPTLC2/3 with the darker shades. A: The orientation of the brown dimer corresponds to that of Figure 8(A), whereas the blue, gray, and green dimers are rotated by, respectively, 90, 180, and 270° in the plane of the sheet and assembled to form a disk. B: Lateral view of the octamer. Compared to the orientation in (A), the disk was rotated 90° anticlockwise along an axis parallel to the plane of the sheet. The four transmembrane helices of the SPTLC1 subunit are represented as in Figure 9. The active site of the green subunit SPTLC2/3 is indicated. Note that the topology of ssSPT, colored red, is unknown.