Enterococcus faecalis mevalonate kinase

Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM.     

Effect of sulfhydryl reagents on mevalonate kinase partially purified from chicken liver

Unlike other vertebrate mevalonate kinase, the enzyme partially purified from neonatal chick liver was not activated by the -SH group protectors reduced glutathione, cysteine, dithiothreitol and beta-mercaptoethanol at any concentrations assayed (0.01-10.00 mM). However, the activity was found to be sensitive to thiol group binding reagents. p-Hydroxymercuribenzoate was the most active inhibitor. At 0.1 mM concentration, p-HMB completely abolished the enzyme activity. N-ethylmaleimide (0.01-1.00 mM) was practically ineffective. Inhibition by p-HMB was temperature dependent, being more potent at 37 degrees C than at 4 degrees C.     

The name's bond......disulfide bond

A repeating theme in the structural biology of disulfide oxidants and isomerases is the extraordinary architectural similarity between functionally related proteins from prokaryotes and eukaryotes. The recently determined structure of full-length yeast protein disulfide isomerase (PDI) reveals a U-shaped molecule with two redox-active sites. It bears a remarkable resemblance to the V-shaped, but dimeric, bacterial disulfide isomerases DsbC and DsbG. Similarly, the much-anticipated structure of the bacterial membrane protein DsbB, the redox partner of DsbA, comprises a flexible redox loop embedded in an antiparallel four-helix bundle. This architecture is similar to that of soluble eukaryotic Ero1p and Erv2p proteins, the redox partners of PDI. Importantly, the DsbB crystal structure is a complex with DsbA, providing our first view of the molecular interactions between these two proteins.     

Weekly oral alendronate in mevalonate kinase deficiency

Background

Mevalonate kinase deficiency (MKD) is caused by mutations in the MVK gene, encoding the second enzyme of mevalonate pathway, which results in subsequent shortage of downstream compounds, and starts in childhood with febrile attacks, skin, joint, and gastrointestinal symptoms, sometimes induced by vaccinations.

Methods

For a history of early-onset corticosteroid-induced reduction of bone mineral density in a 14-year-old boy with MKD, who also had presented three bone fractures, we administered weekly oral alendronate, a drug widely used in the management of osteoporosis and other high bone turnover diseases, which blocks mevalonate and halts the prenylation process.

Results

All of the patient’s MKD clinical and laboratory abnormalities were resolved after starting alendronate treatment.

Conclusions

This observation appears enigmatic, since alendronate should reinforce the metabolic block characterizing MKD, but is crucial because of the ultimate improvement shown by this patient. The anti-inflammatory properties of bisphosphonates are a new question for debate among physicians across various specialties, and requires further biochemical and clinical investigation.

     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.     

Catalysis of protein disulfide bond isomerization in a homogeneous substrate

Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.     

Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily.

The mevalonate-dependent pathway is used by many organisms to synthesize isopentenyl pyrophosphate, the building block for the biosynthesis of many biologically important compounds, including farnesyl pyrophosphate, dolichol, and many sterols. Mevalonate kinase (MVK) catalyzes a critical phosphoryl transfer step, producing mevalonate 5'-phosphate. The crystal structure of thermostable MVK from Methanococcus jannaschii has been determined at 2.4 A, revealing an overall fold similar to the homoserine kinase from M. jannaschii. In addition, the enzyme shows structural similarity with mevalonate 5-diphosphate decarboxylase and domain IV of elongation factor G. The active site of MVK is in the cleft between its N- and C-terminal domains. Several structural motifs conserved among species, including a phosphate-binding loop, have been found in this cavity. Asp(155), an invariant residue among MVK sequences, is located close to the putative phosphate-binding site and has been assumed to play the catalytic role. Analysis of the MVK model in the context of the other members of the GHMP kinase family offers the opportunity to understand both the mechanism of these enzymes and the structural details that may lead to the design of novel drugs.     

二硫键与蛋白质的结构

二硫键是肽链上2个半胱氨酸残基的巯基基团发生氧化反应形成的共价键．具有链内二硫键和链间二硫键2种形式。与氨基酸的氨基氮原子之间形成的稳定共价键不同．二硫键容易被还原而断裂,断裂后可再次氧化重新形成二硫键,因而是可以动态变化的化学键。二硫键是参与一级结构也是形成高级结构的重要化学键,对蛋白质折叠和高级结构的形成与维持十分重要。讨论了二硫键的形成和特征及其与蛋白质结构和功能之间的关系,并讨论了生物学教学中关于二硫键的一些疑问．     

Native disulfide bond formation in proteins

Native disulfide bond formation is critical for the proper folding of many proteins. Recent studies using newly identified protein oxidants, folding catalysts, and mutant cells provide insight into the mechanism of oxidative protein folding in vivo. This insight promises new strategies for more efficient protein production.     

Statistics of disulfide bond formation in proteins

A new set of statistical expressions describing the reformation of disulfide bonds from SH groups is proposed. The results of the statistical calculations of disulfide bond reformation are discussed in terms of protein folding.     

Site-directed labeling of a monoclonal antibody: targeting to a disulfide bond

We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.