Structural, mechanistic and regulatory studies of serine palmitoyltransferase

SLs (sphingolipids) are composed of fatty acids and a polar head group derived from l-serine. SLs are essential components of all eukaryotic and many prokaryotic membranes but S1P (sphingosine 1-phosphate) is also a potent signalling molecule. Recent efforts have sought to inventory the large and chemically complex family of SLs (LIPID MAPS Consortium). Detailed understanding of SL metabolism may lead to therapeutic agents specifically directed at SL targets. We have studied the enzymes involved in SL biosynthesis; later stages are species-specific, but all core SLs are synthesized from the condensation of l-serine and a fatty acid thioester such as palmitoyl-CoA that is catalysed by SPT (serine palmitoyltransferase). SPT is a PLP (pyridoxal 5'-phosphate)-dependent enzyme that forms 3-KDS (3-ketodihydrosphingosine) through a decarboxylative Claisen-like condensation reaction. Eukaryotic SPTs are membrane-bound multi-subunit enzymes, whereas bacterial enzymes are cytoplasmic homodimers. We use bacterial SPTs (e. g. from Sphingomonas) to probe their structure and mechanism. Mutations in human SPT cause a neuropathy [HSAN1 (hereditary sensory and autonomic neuropathy type 1)], a rare SL metabolic disease. How these mutations perturb SPT activity is subtle and bacterial SPT mimics of HSAN1 mutants affect the enzyme activity and structure of the SPT dimer. We have also explored SPT inhibition using various inhibitors (e. g. cycloserine). A number of new subunits and regulatory proteins that have a direct impact on the activity of eukaryotic SPTs have recently been discovered. Knowledge gained from bacterial SPTs sheds some light on the more complex mammalian systems. In the present paper, we review historical aspects of the area and highlight recent key developments.</p

The representation of protein complexes in the Protein Ontology (PRO)

BACKGROUND: Representing species-specific proteins and protein complexes in ontologies that are both human- and machine-readable facilitates the retrieval, analysis, and interpretation of genome-scale data sets. Although existing protin-centric informatics resources provide the biomedical research community with well-curated compendia of protein sequence and structure, these resources lack formal ontological representations of the relationships among the proteins themselves. The Protein Ontology (PRO) Consortium is filling this informatics resource gap by developing ontological representations and relationships among proteins and their variants and modified forms. Because proteins are often functional only as members of stable protein complexes, the PRO Consortium, in collaboration with existing protein and pathway databases, has launched a new initiative to implement logical and consistent representation of protein complexes. DESCRIPTION: We describe here how the PRO Consortium is meeting the challenge of representing species-specific protein complexes, how protein complex representation in PRO supports annotation of protein complexes and comparative biology, and how PRO is being integrated into existing community bioinformatics resources. The PRO resource is accessible at http://pir.georgetown.edu/pro/. CONCLUSION: PRO is a unique database resource for species-specific protein complexes. PRO facilitates robust annotation of variations in composition and function contexts for protein complexes within and between species

Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation

Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d-cycloserine (DCS, also known as Seromycin), is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l-cycloserine (LCS), is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg(378) residue is involved in cycloserine inactivation of SPT

The External Aldimine Form of Serine Palmitoyltransferase: STRUCTURAL, KINETIC, AND SPECTROSCOPIC ANALYSIS OF THE WILD-TYPE ENZYME AND HSAN1 MUTANT MIMICS*

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 Arg378 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

Lip1p: a novel subunit of acyl-CoA ceramide synthase

Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl-CoA-dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity. They copurified with ceramide synthase activity, giving unequivocal evidence that they are subunits of the enzyme. In purified form, the acyl-CoA dependence, fatty acyl-CoA chain length specificity, and Fumonisin B1/Australifungin sensitivity of the ceramide synthase were the same as in cells, showing that these are properties of the enzyme and do not depend upon the membrane environment or other factors. SDS–PAGE analysis of purified ceramide synthase revealed the presence of a novel subunit of the enzyme, Lip1p. Lip1p is a single-span ER membrane protein that is required for ceramide synthesis in vivo and in vitro. The Lip1p regions required for ceramide synthesis are localized within the ER membrane or lumen

Alteration of the function of the UDP-glucuronosyltransferase 1A subfamily by cytochrome P450 3A4: different susceptibility for UGT isoforms and UGT1A1/7 variants.

Permitted by publisher to link to journal article only.Functional protein-protein interactions between UDP-glucuronosyltransferase (UGT)1A isoforms and cytochrome P450 (CYP)3A4 were studied. To this end, UGT1A-catalyzed glucuronidation was assayed in Sf-9 cells that simultaneously expressed UGT and CYP3A4. In the kinetics of UGT1A6-catalyzed glucuronidation of serotonin, both Michaelis constant (Km) and maximal velocity (Vmax) were increased by CYP3A4. When CYP3A4 was coexpressed with either UGT1A1 or 1A7, the Vmax for the glucuronidation of the irinotecan metabolite (SN-38) was significantly increased. S50 and Km both which are the substrate concentration giving 0.5 Vmax were little affected by simultaneous expression of CYP3A4. This study also examined the catalytic properties of the allelic variants of UGT1A1 and 1A7 and their effects on the interaction with CYP3A4. Although the UGT1A1-catalyzing activity of 4-methylumbelliferone glucuronidation was reduced in its variant, UGT1A1*6, the coexpression of CYP3A4 restored the impaired function to a level comparable with the wild type. Similarly, simultaneous expression of CYP3A4 increased the Vmax of UGT1A7*1 (wild type) and *2 (N129K and R131K), whereas the same was not observed in UGT1A7*3 (N129K, R131K, and W208R). In the kinetics involving different concentrations of UDP-glucuronic acid (UDP-GlcUA), the Km for UDP-GlcUA was significantly higher for UGT1A7*2 and *3 than *1. The Km of UGT1A7*1 and *3 was increased by CYP3A4, whereas *2 did not exhibit any such change. These results suggest that (1) CYP3A4 changes the catalytic function of the UGT1A subfamily in a UGT isoform-specific manner and (2) nonsynonymous mutations in UGT1A7*3 reduce not only the ability of UGT to use UDP-GlcUA but also CYP3A4-mediated enhancement of catalytic activity, whereas CYP3A4 is able to restore the UGT1A1*6 function.Australian National Health and Medical Research Council

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with β-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions