HMG CoA Reductase catalyzed the interconversion of a thioester, CoA, and mevalonic acid, key step in isoprenoid pathway, through complex reaction mechanism involving three distinct chemical steps with two molecules cofactor large-scale rearrangements enzyme. Here, we investigate second step, formation thiohemiacetal from mevaldehyde, using time resolved crystallography molecular dynamics (MD) simulations. After triggering by pH jump 6.7 to pH9, carbon-sulfur bond can be observed structures...

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is interest because it represents a family serine-specific protein kinases that lack sequence similarity with all other eukaryotic kinases. Similarity exists instead key motifs prokaryotic histidine and ATPases. 2.5-Å crystal reported here reveals has two domains about the same size. N-terminal half dominated by bundle four amphipathic α-helices, whereas C-terminal folded into an α/β sandwich contains nucleotide-binding...

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

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one two unique domains in the catalytic domain plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form coiled-coil linked by large extended connector loop containing conserved trio aromatic residues. P-CR fit into molecular envelope for derived from small-angle X-ray scattering data. and envelope, combined with homology-based chain trace CesA8 core, were...

Abstract Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg 2+ -decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP ATP, have been shown bind sequentially MDD. Here we report crystals enzyme remains active, allowing visualization conformational changes Enterococcus faecalis MDD that describe sequential steps an induced fit enzymatic...

Abstract HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development novel antibiotics. In this study, we report crystal structures HMGR from Enterococcus faecalis (efHMGR) apo and liganded forms, highlighting several unique features enzyme. Statins, which inhibit human with nanomolar affinity, perform poorly against bacterial homologs. We also potent competitive inhibitor (Chembridge2 ID 7828315 or...

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

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved.However, some of these conserved residues may be used to define different classes for the E2 enzymes.One class can defined by Ubc1 protein, which contains K-65, D-90, and D-120, while corresponding positions Cdc34 (Ubc3) defines a second enzymes, S-73, S-97, S-139, respectively.The presence differences otherwise this family suggests critical specificity function or...

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

Biosynthesis of the isoprenoid precursor, isopentenyl diphosphate, is a critical function in all independently living organisms. There are two major pathways for this synthesis, non-mevalonate pathway found most eubacteria and mevalonate animal cells number pathogenic bacteria. An early step condensation acetyl-CoA acetoacetyl-CoA into HMG-CoA, catalyzed by enzyme HMG-CoA synthase. To explore possibility small molecule inhibitor functioning as non-cell wall antibiotic, structure synthase...

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

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulation of PU-34 cells, a primate bone marrow stromal cell line, resulted in prolonged elevation interleukin-11 (IL-11) mRNA, which can be inhibited by protein synthesis inhibitors.Nuclear run-on assays and actinomycin D experiments demonstrated that the up-regulation IL-11 gene expression is mainly controlled at posttranscriptional level through kinase C (PKC) pathway.Inhibition PKC activity calphostin generated an mRNA degradation intermediate...

Transition state force fields enable MD simulations at the transition of HMGCoA reductase that sample ensemble on μs timescale to identify remote residues affect reaction rate.

The Cdc34 (Ubc3) ubiquitin-conjugating enzyme from Saccharomyces cerevisiae plays an essential role in the progression of cells G1 to S phase cell division cycle. Using a high-copy suppression strategy, we have identified yeast gene (UBS1) whose elevated expression suppresses conditional cycle defects associated with cdc34 mutations. UBS1 encodes 32.2-kDa protein previously unknown function and is identical sequence genomic open reading frame on chromosome II (GenBank accession number...

ABSTRACT HMG-CoA reductase ( Pseudomonas mevalonii ) utilizes mevalonate, coenzyme A (CoA) and the cofactor NAD in a complex mechanism involving two hydride transfers with exchange, accompanied by large conformational changes 50 residue subdomain, to generate HMG-CoA. Details about this such as that allow intermediate formation, exchange product release remain unknown. The formation of proposed intermediates has also not been observed structural studies natural substrates. Having shown be an...

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding complex interplay structure and dynamics large systems that is challenge for both experimental computational approaches. QM/MM methods have been extensively used to study these reactions, but difficulties arising from hybrid treatment system are well documented. More importantly, demands simulations mean reaction can only be considered on timescale nanoseconds even though conformational changes needed react...

<p>Understanding the mechanisms of enzymatic catalysis requires a detailed understanding complex interplay structure and dynamics large systems that is challenge for both experimental computational approaches. QM/MM methods have been extensively used to study these reactions, but difficulties arising from hybrid treatment system are well documented. More importantly, demands simulations mean reaction can only be considered on timescale nanoseconds even though conformational changes...

The emergence of multi-drug resistant pathogenic bacteria is one the great challenges to modern medicine. gram positive cocci Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Enterococcus faecalis (VRE) are two particularly virulent examples. In vivo studies have shown that eukaryotic like 'mevalonate' isoprenoid pathway used by these essential their growth virulence [1]. Our structures HMG-CoA reductase (HMGR) from P. mevalonii demonstrated bacterial enzymes structurally...