Hiroko Ikushiro

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

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 to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395T 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 theSphingomonas 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 α-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.

Acceleration of the Substrate Cα Deprotonation by an Analogue of the Second Substrate Palmitoyl-CoA in Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the pyridoxal 5′-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The binding of l-serine alone to SPT leads to the formation of the external aldimine but does not produce a detectable amount of the quinonoid intermediate. However, the further addition of S-(2-oxoheptadecyl)-CoA, a nonreactive analogue of palmitoyl-CoA, caused the apparent accumulation of the quinonoid. NMR studies showed that the hydrogen-deuterium exchange at Cα of l-serine is very slow in the SPT-l-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. In addition, the observation that the nonreactive palmitoyl-CoA facilitated α-deprotonation indicates that the α-deprotonation takes place before the Claisen-type C–C bond formation, which is consistent with the accepted mechanism of the α-oxamine synthase subfamily enzymes. Structural modeling of both the SPT-l-serine external aldimine complex and SPT-l-serine–palmitoyl-CoA ternary complex suggests a mechanism in which the binding of palmitoyl-CoA to SPT induced a conformation change in the PLP-l-serine external aldimine so that the Cα–H bond of l-serine becomes perpendicular to the plane of the PLP-pyridine ring and is favorable for the α-deprotonation. The model also proposed that the two alternative hydrogen bonding interactions of His159 with l-serine and palmitoyl-CoA play an important role in the conformational change of the external aldimine. This is the unique mechanism of SPT that prevents the formation of the reactive intermediate before the binding of the second substrate.

ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex

Significance The cathelicidin antimicrobial peptide (CAMP) is an innate immune element that promotes antimicrobial defense, but excessive CAMP can stimulate inflammation and tumorigenesis. We recently discovered that external perturbations that induce subtoxic levels of endoplasmic reticulum (ER) stress increase sphingosine-1-phosphate (S1P) production, in turn activating NF-κB–mediated CAMP synthesis. We report here that S1P interacts with the heat shock proteins (HSP90α and GRP94) through a previously unidentified S1P receptor-independent intracellular mechanism, followed by the activation of NF-κB leading to stimulation of CAMP production. These studies illuminate the critical role of both ER stress and S1P in orchestrating stress-specific signals that enhance innate immunity. We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Mechanistic enzymology of serine palmitoyltransferase

Serine palmitoyltransferase, which is one of the α-oxamine synthase family enzymes, catalyzes the condensation reaction of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine, the first and rate-determining step of the sphingolipid biosynthesis. As with other α-oxamine synthase family enzymes, the catalytic reaction is composed of multiple elementary steps, and the mechanism to control these steps to avoid side reactions has been the subject of intensive research in recent years. Combined spectroscopic, kinetic, and structural studies have revealed the finely controlled stereochemical mechanism, in which the His residue conserved among the α-oxamine synthase family enzymes plays a central and critical role. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His159 is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathans conjecture.

Role of a conserved arginine residue during catalysis in serine palmitoyltransferase

SPT binds to SPT by molecular sieving (View interaction)

Expression and Purification of Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis, and catalyzes the PLP-dependent condensation of L-serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. Recently, two different genes, LCB1 and LCB2, have been isolated, and it has been suggested that the SPT enzyme is composed of two subunits named LCBI and LCB2. We constructed a co-expression plasm id for LCBI lacking N-terminal and His6-tagged LCB2 lacking N-terminal. Both proteins were expressed in Escherichia coli and LCBI was co-purified with the His6-tagged LCB2 on metal-immobilized affinity resin. This indicates that LCBI and LCB2 expressed in E. coli form a heterooligomer.

Heme-dependent Inactivation of 5-Aminolevulinate Synthase from Caulobacter crescentus

The biosynthesis of heme is strictly regulated, probably because of the toxic effects of excess heme and its biosynthetic precursors. In many organisms, heme biosynthesis starts with the production of 5-aminolevulinic acid (ALA) from glycine and succinyl-coenzyme A, a process catalyzed by a homodimeric enzyme, pyridoxal 5′-phosphate (PLP)-dependent 5-aminolevulinate synthase (ALAS). ALAS activity is negatively regulated by heme in various ways, such as the repression of ALAS gene expression, degradation of ALAS mRNA, and inhibition of mitochondrial translocation of the mammalian precursor protein. There has been no clear evidence, however, that heme directly binds to ALAS to negatively regulate its activity. We found that recombinant ALAS from Caulobacter crescentus was inactivated via a heme-mediated feedback manner, in which the essential coenzyme PLP was rel eased to form the inactive heme-bound enzyme. The spectroscopic properties of the heme-bound ALAS showed that a histidine-thiolate hexa-coordinated ferric heme bound to each subunit with a one-to-one stoichiometry. His340 and Cys398 were identified as the axial ligands of heme, and mutant ALASs lacking either of these ligands became resistant to heme-mediated inhibition. ALAS expressed in C. crescentus was also found to bind heme, suggesting that heme-mediated feedback inhibition of ALAS is physiologically relevant in C. crescentus.

Structural Biology of Sphingolipid Synthesis

As most eukaryotic enzymes involved in sphingolipid synthesis are membrane-bound proteins analyses of their biochemical and structural are difficult, but sphingolipid-containing bacteria are useful alternatives for enzyme sources. We studied serine palmitoyltransferase (SPT), the first enzyme of the biosynthetic pathway, using this method. To study recombinant SPT’s enzymatic properties, we cloned bacterial SPT genes and then over-expressed them in Escherichia coli and purified the water-soluble enzymes. One yielded crystals good enough for X-ray crystallographic analysis, and the structure of SPT in a complex with the amino acid substrate, L-serine, was successfully determined at 2.3 A resolution. SPT is a homodimer with two pyridoxal 5′-phosphate (PLP) molecules located at the dimer interface. Both subunits contribute side chains to the active sites. The electron density map indicates that a Schiff base is formed between L-serine and PLP in the crystal, so any reaction would stop at the external aldimine intermediate if the co-substrate, palmitoyl-CoA, were absent. Highly conserved amino acids among bacterial and eukaryotic SPTs are also located in the three dimensional structure of this enzyme, and their possible roles in the function of SPT are discussed.