Publications

• Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and other stress responses.

  Perry D.K.

  Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis. As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses. In this review, its role in mediating these resp ... >> More

  Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis. As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses. In this review, its role in mediating these responses is discussed within the context of de novo ceramide synthesis. Furthermore, a discussion is provided of its regulation as discerned from both yeast and mammalian studies. << Less

  Biochim Biophys Acta 1585:146-152(2002) [PubMed] [EuropePMC]

• The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis.

  Nagiec M.M., Baltisberger J.A., Wells G.B., Lester R.L., Dickson R.C.

  The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, b ... >> More

  The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2-to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:7899-7902(1994) [PubMed] [EuropePMC]

• A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction.

  Ikushiro H., Hayashi H., Kagamiyama H.

  Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have ... >> More

  Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme. The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT. << Less

  J. Biol. Chem. 276:18249-18256(2001) [PubMed] [EuropePMC]

• Bacterial serine palmitoyltransferase: a water-soluble homodimeric prototype of the eukaryotic enzyme.

  Ikushiro H., Hayashi H., Kagamiyama H.

  Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimob ... >> More

  Serine palmitoyltransferase (SPT, EC 2.3.1.50) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine and palmitoyl coenzyme A (CoA) to 3-ketodihydrosphingosine (KDS). We found that the gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity, and purified SPT to homogeneity. Unlike eukaryotic enzymes, this enzyme was a water-soluble homodimeric protein. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT showed about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The purified recombinant-SPT showed the characteristic absorption spectrum derived from its coenzyme pyridoxal 5'-phosphate (PLP). The addition of the substrate, L-serine, caused spectral changes indicating the formation of the external aldimine intermediate. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT. << Less

  Biochim Biophys Acta 1647:116-120(2003) [PubMed] [EuropePMC]

• Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities.

  Han G., Gupta S.D., Gable K., Niranjanakumari S., Moitra P., Eichler F., Brown R.H. Jr., Harmon J.M., Dunn T.M.

  Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated t ... >> More

  Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells. << Less

  Proc. Natl. Acad. Sci. U.S.A. 106:8186-8191(2009) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The external aldimine form of serine palmitoyltransferase: structural, kinetic, and spectroscopic analysis of the wild-type enzyme and HSAN1 mutant mimics.

  Raman M.C.C., Johnson K.A., Yard B.A., Lowther J., Carter L.G., Naismith J.H., Campopiano D.J.

  Sphingolipid biosynthesis begins with the condensation of L-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and s ... >> More

  Sphingolipid biosynthesis begins with the condensation of L-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a "dominant" or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg(378) residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity. << Less

  J. Biol. Chem. 284:17328-17339(2009) [PubMed] [EuropePMC]

• Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase.

  Gable K., Han G., Monaghan E., Bacikova D., Natarajan M., Williams R., Dunn T.M.

  It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyl ... >> More

  It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p. << Less

  J. Biol. Chem. 277:10194-10200(2002) [PubMed] [EuropePMC]

• Convergent evolution of bacterial ceramide synthesis.

  Stankeviciute G., Tang P., Ashley B., Chamberlain J.D., Hansen M.E.B., Coleman A., D'Emilia R., Fu L., Mohan E.C., Nguyen H., Guan Z., Campopiano D.J., Klein E.A.

  The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined s ... >> More

  The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homolog. Here we used genomic and biochemical approaches to identify six proteins comprising the complete pathway for bacterial ceramide synthesis. Bioinformatic analyses revealed the widespread potential for bacterial ceramide synthesis leading to our discovery of a Gram-positive species that produces ceramides. Biochemical evidence demonstrated that the bacterial pathway operates in a different order from that in eukaryotes. Furthermore, phylogenetic analyses support the hypothesis that the bacterial and eukaryotic ceramide pathways evolved independently. << Less

  Nat. Chem. Biol. 18:305-312(2022) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.