Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1- phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping con- firms functional involvement of binding site residues. A p helix (p bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, a-helically dominated, ‘‘sandwich’’ topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes

\u3ci\u3eArabidopsis\u3c/i\u3e Accelerated Cell Death 11, ACD11, Is a Ceramide-1-Phosphate Transfer Protein and Intermediary Regulator of Phytoceramide Levels

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1- phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, ‘‘sandwich’’ topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily

Phosphatidylserine stimulates ceramide 1-phosphate (C1P) intermembrane transfer by C1P transfer proteins

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, “soluble” phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium

In the cell, propionate is mainly formed during \beta-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that L-threonine is non-oxidatively cleaved to propionate via 2-ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.-). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group $P3_{1}21$ or $P3_{2}21$, with unit-cell parameters a = b = 111.47, c = 66.52 Angstron…. A complete data set to 2.2 Angstron… resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4AA{p_4}A: Evidence for a novel Ap4AA{p_4}A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine $5^{\prime}$, $5^{{\prime}{\prime}{\prime}}-{P^1}$, $P^4$-tetraphosphate $(A{p_4}A)$ by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed $Ap_4A$ bound to the active site pocket suggesting the presence of $Ap_4A$ synthetic activity in TdcD. Binding of $Ap_4A$ to the enzyme was confirmed by the structure determination of a TdcD-$A{p_4}A$ complex obtained after cocrystallization of TdcD with commercially available $A{p_4}A$. Mass spectroscopic studies provided further evidence for the formation of $A{p_4}A$ by propionate kinase in the presence of ATP. In the TdcD-$A{p_4}A$ complex structure, $A{p_4}A$ is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction

Crystal structures of ADP and AMPPNP-bound Propionate kinase (TdcD)from Salmonella typhimurium: Comparison with Members of Acetate and Sugar kinase/Heat Shock Cognate 70/Actin Superfamily

Recently, it has been shown that L-threonine can be catabolized non-oxidatively to propionate via 2-ketobutyrate. Propionate kinase(TdcD; EC 2.7.2.-) catalyses the last step of this metabolic process by enabling the conversion of propionyl phosphate and ADP to propionate and ATP. To provide insights into the substrate-binding pocket and catalytic mechanism of TdcD, the crystal structures of the enzyme from Salmonella typhimurium in complex with ADP and AMPPNP have been determined to resolutions of 2.2 angstrom and 2.3 angstrom,respectively, by molecular replacement using Methanosarcina thermophilaacetate kinase (MAK; EC 2.7.2.1). Propionate kinase, like acetate kinase, contains a fold with the topology \beta\beta\beta\alpha\beta\alpha\beta\alpha identical with that of glycerol kinase, hexokinase,heat shock cognaten 70 (Hsc70) and actin, the superfamily of phosphotransferases. The structure consists of two domains with the active site contained in a cleft at the domain interface. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate.This was further confirmed by kinetic studies with the purified enzyme,which showed about ten times lower $K_{m}$ for propionate (2.3 mM) than foracetate (26.9 mM). Comparison of TdcD complex structures with those of acetate and sugar kinase/Hsc70/actin obtained with different ligands has permitted the identification of catalytically essential residues involved in substrate binding and catalysis, and points to both structural and mechanistic similarities. In the well characterized members of this superfamily, ATP phosphoryl transfer or hydrolysis is coupled to a large conformational change in which the two domains close around the active site cleft. The significant amino acid sequence similarity between TdcD and MAK has facilitated study of domain movement, which indicates that the conformation assumed by the two domains in the nucleotide-bound structure of TdcD may represent an intermediate point in the pathway of domain closure

RAS Proteins and Their Regulators in Human Disease

RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. These switches are normally tightly controlled, but in RAS-related diseases, such as cancer, RASopathies, and many psychiatric disorders, mutations in the RAS genes or their regulators render RAS proteins persistently active. The structural basis of the switch and many of the pathways that RAS controls are well known, but the precise mechanisms by which RAS proteins function are less clear. All RAS biology occurs in membranes: a precise understanding of RAS' interaction with membranes is essential to understand RAS action and to intervene in RAS-driven diseases

Structure of the putative mutarotase YeaD from Salmonella typhimurium in two different forms: in vivo binding of a sugar phosphate

Salmonella typhimurium YeaD (stYeaD), annotated as a putative aldose 1-epimerase, has a very low sequence identity to other well characterized mutarotases. Sequence analysis suggested that the catalytic residues and a few of the substrate-binding residues of galactose mutarotases (GalMs) are conserved in stYeaD. Determination of the crystal structure of stYeaD in an orthorhombic form at 1.9 angstrom resolution and in a monoclinic form at 2.5 angstrom resolution revealed this protein to adopt the beta-sandwich fold similar to GalMs. Structural comparison of stYeaD with GalMs has permitted the identification of residues involved in catalysis and substrate binding. In spite of the similar fold and conservation of catalytic residues, minor but significant differences were observed in the substrate- binding pocket. These analyses pointed out the possible role of Arg74 and Arg99, found only in YeaD-like proteins, in ligand anchoring and suggested that the specificity of stYeaD may be distinct from those of GalM