Peripheral Membrane Enzyme Research Articles

Architecture and function of yeast phosphatidate phosphatase Pah1 domains/regions

Phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, provides a direct precursor for the synthesis of the storage lipid triacylglycerol and the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The enzyme controlling the key phospholipid PA also plays a crucial role in diverse aspects of lipid metabolism and cell physiology. PA phosphatase is a peripheral membrane enzyme that is composed of multiple domains/regions required for its catalytic function and subcellular localization. In this review, we discuss the domains/regions of PA phosphatase from the yeast Saccharomyces cerevisiae with reference to the homologous enzyme from mammalian cells.

Corynebacterium glutamicum pyruvate:quinone oxidoreductase: an enigmatic metabolic enzyme with unusual structural features.

Pyruvate:quinone oxidoreductase (PQO) is a flavin-containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate-medium. A similar pattern with about 30-fold higher specific PQO activities was observed in C. glutamicum with plasmid-bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post-transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h-1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate-medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein-1) and turnover number (kcat: 440 vs. 78 s-1, respectively), suggesting post-translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C-terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C-terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C-terminus for enzyme activation and lipid binding.

SPLA2 Wobbles on the Lipid Bilayer between Three Positions, Each Involved in the Hydrolysis Process.

Secreted phospholipases A2 (sPLA2s) are peripheral membrane enzymes that hydrolyze phospholipids in the sn-2 position. The action of sPLA2 is associated with the work of two active sites. One, the interface binding site (IBS), is needed to bind the enzyme to the membrane surface. The other one, the catalytic site, is needed to hydrolyze the substrate. The interplay between sites, how the substrate protrudes to, and how the hydrolysis products release from, the catalytic site remains in the focus of investigations. Here, we report that bee venom PLA2 has two additional interface binding modes and enzyme activity through constant switching between three different orientations (modes of binding), only one of which is responsible for substrate uptake from the bilayer. The finding was obtained independently using atomic force microscopy and molecular dynamics. Switching between modes has biological significance: modes are steps of the enzyme moving along the membrane, product release in biological milieu, and enzyme desorption from the bilayer surface.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains

Significance A cell must be able to measure whether the lipid membranes that surround its insides are stretched. Currently, mechanosensitive ion channels are the best-studied class of membrane tension sensors, but recent work suggests that peripheral membrane enzymes that gauge nuclear confinement or swelling during cell migration or upon tissue injury constitute a second class. The mechanosensitivity of these enzymes derives from their calcium-dependent (“C2-like”) membrane-interaction domains. Although these can be found in many important signaling proteins, they have remained virtually unstudied as mechanotransducers. How membrane tension controls these domains and what features render them mechanosensitive is unclear. Here, we show that membrane tension-sensing by C2-like domains is mediated by a synergy between mechanosensitive calcium-binding and membrane insertion.

The phosphatidic acid pathway enzyme PlsX plays both catalytic and channeling roles in bacterial phospholipid synthesis

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (βα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein-lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation-π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

Computational Analysis of LOX1 Inhibition Identifies Descriptors Responsible for Binding Selectivity.

Lipoxygenases are a family of cytosolic, peripheral membrane enzymes, which catalyze the hydroperoxidation of polyunsaturated fatty acids and are implicated in the pathogenesis of major human diseases. Over the years, a substantial number of scientific reports have introduced inhibitors active against one or another subtype of the enzyme, but the selectivity issue has proved to be a major challenge for drug design. In the present work, we assembled a dataset of 317 structurally diverse molecules hitherto reported as active against 15S-LOX1, 12S-LOX1, and 15S-LOX2 and identified, using supervised machine learning, a set of structural descriptors responsible for the binding selectivity toward the enzyme 15S-LOX1. We subsequently incorporated these descriptors in the training of QSAR models for LOX1 activity and selectivity. The best performing classifiers are two stacked models that include an ensemble of support vector machine, random forest, and k-nearest neighbor algorithms. These models not only can predict LOX1 activity/inactivity but also can discriminate with high accuracy between molecules that exhibit selective activity toward either one of the isozymes 15S-LOX1 and 12S-LOX1.

The quinone-binding site of Acidithiobacillus ferrooxidans sulfide: quinone oxidoreductase controls both sulfide oxidation and quinone reduction.

Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane enzyme that catalyzes the oxidation of sulfide and the reduction of ubiquinone. Ubiquinone binds to a conserved hydrophobic domain and shuttles electrons from a noncovalent flavin adenine dinucleotide cofactor to the membrane-bound quinone pool. Utilizing the structure of decylubiquinone bound to Acidithiobacillus ferrooxidans SQR, we combined site-directed mutagenesis and kinetic approaches to analyze quinone binding. SQR can reduce both benzoquinones and naphthoquinones. The alkyl side-chain of ubiquinone derivatives enhances binding to SQR but limits the enzyme turnover. Pentachlorophenol and 2-n-heptyl-4-hydroxyquinoline-N-oxide are potent inhibitors of SQR with apparent inhibition constants (Ki) of 0.46 μmol·L(-1) and 0.58 μmol·L(-1), respectively. The highly conserved amino acids surrounding the quinone binding site play an important role in quinone reduction. The phenyl side-chains of Phe357 and Phe391 sandwich the benzoquinone head group and are critical for quinone binding. Importantly, conserved amino acids that define the ubiquinone-binding site also play an important role in sulfide oxidation/flavin reduction.

Substrate Uptake and Subcellular Compartmentation of Anoxic Cholesterol Catabolism in Sterolibacterium denitrificans

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. (13)C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a β-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C27 sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C27 acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.

Tunable Nucleation Time of Functional Sphingomyelinase–Lipid Features Studied by Membrane Array Statistic Tool

Aggregation or assembly of lipids and proteins could significantly change the proteins' function. A peripheral membrane enzyme, sphingomyelinase (SMase), has been reported to be able to assemble to a functional feature with its lipid substrate, sphingomyelin (SM), and its lipid product, ceramide (Cer). SMase seems to processes its substrate more effectively in this feature. Here, we report that the functional feature has a tunable formation time. The peculiar behavior is that the feature formation has a time lag depending on the membrane composition. We hypothesized that the time lag is due to the significant nucleation energy barrier when the feature phase forms in its metastable parent phase in the 2-D lipid membrane. To study the stochastic nucleation of the feature, we built a corralled lipid membrane platform with numerous isolated membrane systems in parallel to capture the nucleation statistics. Using the high-throughput approach and the appropriate experimental design to circumvent the interplay of the complicated phase segregation in membranes induced by SMase, we found that the nucleation rate of the feature can be tuned by the supersaturation of the enzyme, the lipid substrate, and the lipid product, in the fluid phase of the membrane. The correlation between the supersaturation and the nucleation rate can be well described by the classical nucleation theory equation, suggesting that the feature formation follows the nucleation process with a certain component ratio specified in the equation. The certain relative component ratio suggests that the feature may have certain organization instead of being random aggregation. In addition, our finding suggests that nucleation could serve as a time lag control mechanism in this enzymatic system, and ways to reduce nucleation energy barrier could be used to shorten the aggregation time lag and vice versa.

LpxI: A Novel Phosphosaccharolipid Hydrolase

Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids, while the outer leaflet contains mainly lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. LPS is anchored to the outer membrane by lipid A, a unique glucosamine-based saccharolipid. Lipid A biosynthesis is required for bacterial viability and pathogenesis. While most lipid A biosynthetic genes are present in a single copy, one gene, lpxH, encoding a membrane-associated specific UDP-diacylglucosamine hydrolase, is absent in ∼30% of Gram-negative bacteria. We hypothesized that a transformational analogue of lpxH must exist in these organisms. We identified this gene, designated lpxI, in Caulobacter crescentus, and confirmed its ability to cover for a deficiency of lpxH in Eschserichia coli. LpxI lacks homology to any other known enzyme. We over-expressed LpxI, purified it, and obtained protein crystals. We solved the X-ray crystal structure of this peripheral membrane enzyme using a single-wavelength anomalous dispersion (SAD) dataset collected on Se-Met derivatized protein. The structural data reveal two domains, each having a novel fold. Unexpectedly, “apo” LpxI co-purified and co-crystallized stoichiometrically with its product, diacylglucosamine-1-phosphate (lipid X), the saccharolipid precursor of lipid A. We then identified and determined the 2.5 A X-ray crystal structure of an inactive point mutant of Caulobacter crescentus LpxI, which co-purified in a stoichiometric ratio with its substrate, UDP-2,3-diacyglucosamine. The conformation of substrate-liganded LpxI differs significantly from the product -liganded wild-type enzyme. Taken together with analytical unltracentrifugation data, this observation suggests that large-scale domain re-arrangement occurs during LpxI substrate binding and/or catalysis. These data provide an interesting example of lipid-protein interaction, and set the stage for additional structural and mechanistic work.

Production and purification of the penicillin-binding protein 3 from Pseudomonas aeruginosa

The penicillin-binding proteins (PBPs) are peripheral membrane enzymes that catalyze the final steps for the biosynthesis of the essential bacterial cell wall heteropolymer peptidoglycan. Bacteria produce a number and variety of PBPs which are classified as either high molecular weight or low molecular weight PBPs. The high molecular weight PBPs are multimodular being comprised of an N-terminal membrane anchor followed by a non-pencillin binding domain and a C-terminal penicillin-binding domain. The penicillin-binding domain functions as a serine-acyl transpeptidase to catalyze the crosslinking of neighboring glycan strands within the peptidoglycan sacculus. PBP 3 from Escherichia coli has been studied extensively and it has been shown to be responsible for the synthesis of peptidoglycan during the division and septation of the cells. The opportunistic human pathogen Pseudomonas aeruginosa produces a similar compliment of PBPs to E. coli, but differences in their organization and function have been noted. To investigate these differences further, appropriate quantities of each of the P. aeruginosa PBPs are required in forms amenable for study both in vivo and in vitro. Herein, we describe the cloning and expression of the ftsI gene encoding PBP 3from P. aeruginosa. The PBP was engineered in soluble form to facilitate its study in vitro and with a hexa-His tag to permit its facile purification by affinity chromatography. The recombinant proteins were demonstrated to bind penicillin and these forms of the PBPs were shown to be useful in studying their localization within their host cells by immunogold transmission electron microscopy.

Discovery, characterization, and structural determination of a novel UDP‐2,3‐diacylglucosamine hydrolase

Gram‐negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids, while the outer leaflet contains mainly lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram‐negative bacteria from antibiotics and other environmental stressors. LPS is anchored to the outer membrane by Lipid A, a unique glucosamine‐based saccharolipid. Lipid A biosynthesis is required for viability and pathogenesis. While most lipid A biosynthetic genes are present in a single copy, one gene, lpxH, encoding a membrane‐associated specific UDP‐diacylglucosamine hydrolase, is absent in ~30% of Gram‐negative bacteria. We hypothesized that a transformational analogue of lpxH must exist in these organisms. We identified this gene, designated lpxI, in Caulobacter crescentus, and confirmed its ability to cover for a deficiency of lpxH in Eschserichia coli. LpxI lacks homology to any other known enzyme. We over‐expressed LpxI, purified it, and obtained protein crystals. We solved the x‐ray crystal structure of this peripheral membrane enzyme to a resolution of 2.7 angstroms using a single‐wavelength anomalous dispersion (SAD) dataset collected on Se‐Met derivatized protein. The structural data reveal two domains, each having a novel fold. Unexpectedly, “apo” LpxI co‐purified and co‐crystallized in a 1:1 stoichiometric ratio with its product, lipid X. These data provide an interesting example of lipid‐protein interaction, and set the stage for additional structural and mechanistic work. Support was provided by NIH grant GM5150 to C.R.H.R., Specialized Center grant of the Protein Structure Initiative U54 GM074929‐01 to R.M.S., and NSF GRF grant 2005029353 to L.E.M.

Reaction mechanisms of thiamin diphosphate enzymes: redox reactions

Amongst a wide variety of different biochemical reactions in cellular carbon metabolism, thiamin diphosphate-dependent enzymes catalyze the oxidative decarboxylation of 2-keto acids. This type of reaction typically involves redox coupled acyl transfer to CoA or phosphate and is mediated by additional cofactors, such as flavins, iron-sulfur clusters or lipoamide swinging arms, which transmit the reducing equivalents that arise during keto acid oxidation to a final electron acceptor. EPR spectroscopic and kinetic studies have implicated the intermediacy of radical cofactor intermediates in pyruvate:ferredoxin oxidoreductase and an acetyl phosphate-producing pyruvate oxidase, whereas the occurrence of transient on-pathway radicals in other enzymes is less clear. The structures of pyruvate:ferredoxin oxidoreductase and pyruvate oxidase with different enzymic reaction intermediates along the pathway including a radical intermediate were determined by cryo-crystallography and used to infer electron tunneling pathways and the potential roles of CoA and phosphate for an intimate coupling of electron and acyl group transfer. Viable mechanisms of reductive acetylation in pyruvate dehydrogenase multi-enzyme complex, and of electron transfer in the peripheral membrane enzyme pyruvate oxidase from Escherichia coli, are also discussed.

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO(2) and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles.

Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase fromEscherichia coli

The thiamine diphosphate- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Delta23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (full-length EcPOX) or 2-methyl-2,4-pentanediol (Delta23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 A. The two forms of EcPOX crystallize in different space groups. Whereas full-length EcPOX crystallizes in the tetragonal space group P4(3)2(1)2 with two monomers per asymmetric unit, the crystals of Delta23 EcPOX belong to the orthorhombic space group P2(1)2(1)2(1) and contain 12 monomers per asymmetric unit.

Effects of extremely low frequency electromagnetic fields on membrane-associated enzymes

The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54–61% with field amplitudes above a threshold of 73–151 μT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.

Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix.

sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer. For it to display its enzymic activity, binding to the inner membrane is required. The way the enzyme interacts with the membrane and how this controls activity has not been elucidated. In the present study we provide evidence for direct protein-lipid interaction. Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids. GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers. From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids.

Electrochemical and Surface Plasmon Resonance Characterization of the Step-by-Step Self-Assembly of a Biomimetic Structure onto an Electrode Surface

A plane gold-supported bilayer was prepared on an electrode by fusion of phospholipid (dimyristoylphosphatidylcholine (DMPC)) vesicles onto an alkanethiol (octadecylmercaptan (OM)) self-assembled monolayer (SAM). Escherichia coli pyruvate oxidase (Pox), a peripheral membrane enzyme, was incorporated into the supported bilayer. This supramolecular assembly was characterized by contact angle goniometry, electrochemical blocking studies, double-layer capacitance, and BIAlite (surface plasmon resonance) measurements. Electrochemistry of ferrocenemethanol at the gold surface was blocked by the well-ordered alkane chains of the OM monolayer. In order to prevent this blocking effect, dibenzyl disulfide (DBDS) was used to produce defect sites in the OM monolayer and to allow the reversibility of ferrocene electrochemistry. BIAlite measurements showed that fusion of DMPC on the OM + DBDS monolayer was not significantly different from the fusion of DMPC on the OM monolayer. Pox incorporation into the (OM + DBDS)/DM...