Synthesis of Caged HMG-CoA Reductase Substrates for the Elucidation of Cellular Pathways.

The synthesis of photocaged substrates of the biologically important enzyme HMG-CoA reductase is reported. HMG-CoA bearing a -hydroxyphenacyl (pHP) photocage moiety was synthesized in an overall yield of 14% over seven steps in addition to caged forms of mevalonate and mevaldehyde. The absorption maximum and quantum yield for the decaging of the photocaged compounds are dependent on pH with a λ of 330 nm and a ϕ of 5%, respectively, at pH 9.

pH-dependent reaction triggering in PmHMGR crystals for time-resolved crystallography.

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD. The multi-step reaction mechanism involves an exchange of bound NAD and large conformational changes by a 50-residue subdomain.

Computational Study of Base-Catalyzed Thiohemiacetal Decomposition in HMG-CoA Reductase.

Thiohemiacetals are key intermediates in the active sites of many enzymes catalyzing a variety of reactions. In the case of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), this intermediate connects the two hydride transfer steps where a thiohemiacetal is the product of the first hydride transfer and its breakdown forms the substrate of the second one, serving as the intermediate during cofactor exchange. Despite the many examples of thiohemiacetals in a variety of enzymatic reactions, there are few studies that detail their reactivity.

Heterogeneity in Lowe Syndrome: Mutations Affecting the Phosphatase Domain of OCRL1 Differ in Impact on Enzymatic Activity and Severity of Cellular Phenotypes.

Lowe Syndrome (LS) is a condition due to mutations in the gene, characterized by congenital cataracts, intellectual disability, and kidney malfunction. Unfortunately, patients succumb to renal failure after adolescence. This study is centered in investigating the biochemical and phenotypic impact of patient's OCRL1 variants (OCRL1).

Targeting Enterococcus faecalis HMG-CoA reductase with a non-statin inhibitor.

HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development of novel antibiotics. In this study, we report the crystal structures of HMGR from Enterococcus faecalis (efHMGR) in the apo and liganded forms, highlighting several unique features of this enzyme. Statins, which inhibit the human enzyme with nanomolar affinity, perform poorly against the bacterial HMGR homologs.

Essential amino acids in the Plant-Conserved and Class-Specific Regions of cellulose synthases.

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant.

Microsecond timescale MD simulations at the transition state of HMGR predict remote allosteric residues.

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to reach the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale.

Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase.

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction.

Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 μM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen .

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from (MDD).

The nuclear mitotic apparatus protein NuMA controls rDNA transcription and mediates the nucleolar stress response in a p53-independent manner.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined.

Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance.

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX.

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates.

We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication.

In vitro reassembly of the ribose ATP-binding cassette transporter reveals a distinct set of transport complexes.

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate.

The increasingly complex mechanism of HMG-CoA reductase.

HMG-CoA reductase (HMGR) is the target of statins, cholesterol-lowering drugs prescribed to millions of patients worldwide. More recent research indicates that HMGR could be a useful target in the development of antimicrobial agents. Over the last seven decades, researchers have proposed a series of increasingly complex reaction mechanisms for this biomedically important enzyme.

A novel role for coenzyme A during hydride transfer in 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation-reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations.

Crystallographic analysis of the ENTH domain from yeast epsin Ent2 that induces a cell division phenotype.

Epsins are eukaryotic, endocytic adaptor proteins primarily involved in the early steps of clathrin mediated endocytosis. Two epsins exist in Saccharomyces cerevisiae, Ent1 and Ent2, with single epsin knockouts being viable, while the double knockout is not. These proteins contain a highly conserved Epsin N-terminal homology (ENTH) domain that is essential for cell viability.

Molecular modeling of the reaction pathway and hydride transfer reactions of HMG-CoA reductase.

HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance because of the impact of its statin inhibitors on public health. Although the reaction has been studied extensively using X-ray crystallography, there are surprisingly no computational studies that test the mechanistic hypotheses suggested for this complex reaction. Theozyme and quantum mechanical (QM)/molecular mechanical (MM) calculations up to the B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p) level of theory were employed to generate an atomistic description of the enzymatic reaction process and its energy profile.

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring.

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP.

Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry.

Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring.

Identification of novel inhibitors for a low molecular weight protein tyrosine phosphatase via virtual screening.

The human cytoplasmic protein tyrosine phosphatase (HCPTP) has been identified as a potential target for inhibition in order to downregulate metastatic transformation in several human epithelial cancers such as breast, prostate and colon cancer. Docking with two scoring functions on both isoforms of HCPTP was employed as an initial virtual screen to identify potential inhibitors. Compounds identified as potential inhibitors via this in silico screen were subjected to kinetic analysis in order to validate their selection as improved inhibitors.

A structural limitation on enzyme activity: the case of HMG-CoA synthase.

Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine.

Expression and purification of the intact cytoplasmic domain of the human ephrin receptor A2 tyrosine kinase in Escherichia coli.

The ephrin receptor A2 (EphA2) is an integral membrane protein tyrosine kinase and a member of the Eph family, the largest known family of receptor tyrosine kinases. EphA2 overexpression is sufficient to transform normal epithelial cells into an aggressive, metastatic phenotype. In normal cells, EphA2 negatively regulates cell growth and invasiveness.

Crystal structure of the human B-form low molecular weight phosphotyrosyl phosphatase at 1.6-A resolution.

The crystal structure of HPTP-B, a human isoenzyme of the low molecular weight phosphotyrosyl phosphatase (LMW PTPase) is reported here at a resolution of 1.6 A. This high resolution structure of the second human LMW PTPase isoenzyme provides the opportunity to examine the structural basis of different substrate and inhibitor/activator responses.