Antoni R. Slabas

Molecular basis of triclosan activity.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) has been used for more than 30 years as a general antibacterial and antifungal agent, and is found in formulations as diverse as toothpastes, cosmetics, antiseptic soaps, carpets, plastic kitchenware and toys. It has recently been suggested that triclosan blocks lipid biosynthesis by specifically inhibiting the enzyme enoyl-acyl carrier protein reductase (ENR). We have carried out a structural analysis and inhibition experiments on a complex of ENR from the bacterium Escherichia coli with triclosan and NAD+. We find that triclosan acts as a site-directed, very potent inhibitor of the enzyme by mimicking its natural substrate.

Proteomic analysis of the Arabidopsis thaliana cell wall

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two‐dimensional gel electrophoresis (2‐DE). The proteins were identified by peptide mass fingerprinting using matrix‐assisted laser desorption/ionisation‐time of flight (MALDI‐TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.

A mechanism of drug action revealed by structural studies of enoyl reductase.

Enoyl reductase (ENR), an enzyme involved in fatty acid biosynthesis, is the target for antibacterial diazaborines and the front-line antituberculosis drug isoniazid. Analysis of the structures of complexes of Escherichia coli ENR with nicotinamide adenine dinucleotide and either thienodiazaborine or benzodiazaborine revealed the formation of a covalent bond between the 2′ hydroxyl of the nicotinamide ribose and a boron atom in the drugs to generate a tight, noncovalently bound bisubstrate analog. This analysis has implications for the structure-based design of inhibitors of ENR, and similarities to other oxidoreductases suggest that mimicking this molecular linkage may have generic applications in other areas of medicinal chemistry.

Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose–hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with β,γ-methyleneadenosine 5′-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

The biochemistry and molecular biology of plant lipid biosynthesis

In the areas of cell biology, biophysics and the biochemistry of signal perception and transduction major advances have come about from an appreciation of the importance of biological membranes. Such membranes not only provide a barrier to the outside of the cell but also define the limits of subcellular organelles contained within them. Subcellular organelles have been fractionated and biochemical compartmentalization of enzyme function deduced by classical work of de Duve and colleagues [16]. Initial electron microscopy observations concentrated on the lipid bilayer description of a biological membrane [18]. Subsequent studies by Singer and Nicholson [114] have led to an appreciation of the importance of the proteins in membranes resulting in the fluid mosaic model. The importance of biological membranes has been additionally highlighted with the development of the chemiosmotic hypothesis of energy generation in biological systems.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Microtubules, protoplasts and plant cell shape : An immunofluorescent study.

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 and 2 Regulate Phospholipid Synthesis at the Endoplasmic Reticulum in Arabidopsis

Regulation of membrane biogenesis is important for cell function. In this article, two phosphatic acid phosphohydrolase enzymes from Arabidopsis are characterized, and it is shown that their disruption leads to activation of phospholipid synthesis and altered endoplasmic reticulum membrane morphology. The data suggest that either the enzymes or their substrate/product regulate endoplasmic reticulum membrane biogenesis. Phospholipid biosynthesis is essential for the construction of most eukaryotic cell membranes, but how this process is regulated in plants remains poorly understood. Here, we show that in Arabidopsis thaliana, two Mg2+-dependent phosphatidic acid phosphohydrolases called PAH1 and PAH2 act redundantly to repress phospholipid biosynthesis at the endoplasmic reticulum (ER). Leaves from pah1 pah2 double mutants contain ~1.8-fold more phospholipid than the wild type and exhibit gross changes in ER morphology, which are consistent with massive membrane overexpansion. The net rate of incorporation of [methyl-14C]choline into phosphatidylcholine (PC) is ~1.8-fold greater in the double mutant, and the transcript abundance of several key genes that encode enzymes involved in phospholipid synthesis is increased. In particular, we show that PHOSPHORYLETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1) is upregulated at the level of transcription in pah1 pah2 leaves. PEAMT catalyzes the first committed step of choline synthesis in Arabidopsis and defines a variant pathway for PC synthesis not found in yeasts or mammals. Our data suggest that PAH1/2 play a regulatory role in phospholipid synthesis that is analogous to that described in Saccharomyces cerevisiae. However, the target enzymes differ, and key components of the signal transduction pathway do not appear to be conserved.

Common themes in redox chemistry emerge from the X-ray structure of oilseed rape (Brassica napus) enoyl acyl carrier protein reductase.

BACKGROUND Enoyl acyl carrier protein reductase (ENR) catalyzes the NAD(P)H-dependent reduction of trans-delta 2-enoyl acyl carrier protein, an essential step in de novo fatty acid biosynthesis. Plants contain both NADH-dependent and separate NADPH-dependent ENR enzymes which form part of the dissociable type II fatty acid synthetase. Highly elevated levels of the NADH-dependent enzyme are found during lipid deposition in maturing seeds of oilseed rape (Brassica napus). RESULTS The crystal structure of an ENR-NAD binary complex has been determined at 1.9 A resolution and consists of a homotetramer in which each subunit forms a single domain comprising a seven-stranded parallel beta sheet flanked by seven alpha helices. The subunit has a topology highly reminiscent of a dinucleotide-binding fold. The active site has been located by difference Fourier analysis of data from crystals equilibrated in NADH. CONCLUSIONS The structure of ENR shows a striking similarity with the epimerases and short-chain alcohol dehydrogenases, in particular, 3 alpha,20 beta-hydroxysteroid dehydrogenase (HSD). The similarity with HSD extends to the conservation of a catalytically important lysine that stabilizes the transition state and to the use of a tyrosine as a base--with subtle modifications arising from differing requirements of the reduction chemistry.

The X-ray structure of Brassica napus β-keto acyl carrier protein reductase and its implications for substrate binding and catalysis

BACKGROUND beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.