An Coupled Assay for PEPC with Control of Bicarbonate Concentration.

Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C compound oxaloacetate. This enzyme is particularly important in the context of C photosynthesis, where it is the initial carbon-fixing enzyme. Many studies have used kinetic approaches to characterize the properties of PEPCs from different species, different post-translational states, and after mutagenesis.

The active site of magnesium chelatase.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology.

Kinetic Modifications of C PEPC Are Qualitatively Convergent, but Larger in Than in .

C photosynthesis results from a set of anatomical features and biochemical components that act together to concentrate CO within the leaf and boost productivity. This complex trait evolved independently many times, resulting in various realizations of the phenotype, but in all C plants the primary fixation of atmospheric carbon is catalyzed by phosphoenolpyruvate carboxylase. Comparisons of C and non-C PEPC from a few closely related species suggested that the enzyme was modified to meet the demands of the C cycle.

Lateral Gene Transfer Acts As an Evolutionary Shortcut to Efficient C4 Biochemistry.

The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function.

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase.

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP-dependent insertion of a Mg ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion.

The coproporphyrin ferrochelatase of : mechanistic insights into a regulatory iron-binding site.

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from and are known to be inhibited by elevated iron concentrations.

The catalytic power of magnesium chelatase: a benchmark for the AAA(+) ATPases.

In the first committed reaction of chlorophyll biosynthesis, magnesium chelatase couples ATP hydrolysis to the thermodynamically unfavorable Mg(2+) insertion into protoporphyrin IX (ΔG°' of circa 25-33 kJ·mol(-1) ). We explored the thermodynamic constraints on magnesium chelatase and demonstrate the effect of nucleotide hydrolysis on both the reaction kinetics and thermodynamics. The enzyme produces a significant rate enhancement (kcat /kuncat of 400 × 10(6) m) and a catalytic rate enhancement, kcat/KmDIXK0.

Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus.

Characterization of the magnesium chelatase from Thermosynechococcus elongatus.

The first committed step in chlorophyll biosynthesis is catalysed by magnesium chelatase (E.C. 6.

The allosteric role of the AAA+ domain of ChlD protein from the magnesium chelatase of synechocystis species PCC 6803.

Magnesium chelatase is an AAA(+) ATPase that catalyzes the first step in chlorophyll biosynthesis, the energetically unfavorable insertion of a magnesium ion into a porphyrin ring. This enzyme contains two AAA(+) domains, one active in the ChlI protein and one inactive in the ChlD protein. Using a series of mutants in the AAA(+) domain of ChlD, we show that this site is essential for magnesium chelation and allosterically regulates Mg(2+) and MgATP(2-) binding.

Nonequilibrium isotope exchange reveals a catalytically significant enzyme-phosphate complex in the ATP hydrolysis pathway of the AAA(+) ATPase magnesium chelatase.

Magnesium chelatase is an AAA(+) ATPase that catalyzes the first committed step in chlorophyll biosynthesis. Using nonequilibrium isotope exchange, we show that the ATP hydrolysis reaction proceeds via an enzyme-phosphate complex. Exchange from radiolabeled phosphate to ATP was not observed, offering no support for an enzyme-ADP complex.

Burkholderia gladioli - a predictor of poor outcome in cystic fibrosis patients who receive lung transplants? A case of locally invasive rhinosinusitis and persistent bacteremia in a 36-year-old lung transplant recipient with cystic fibrosis.

There have been very few reports describing postlung transplant outcomes in patients' infected⁄colonized with Burkholderia gladioli pretransplant. A case involving a lung transplant recipient with cystic fibrosis who ultimately died as a result of severe rhinosinusitis due to B gladioli infection in the context of postlung transplant immunosuppression is reported.

A case of adalimumab-induced pneumonitis in a 45-year-old man with Crohn's disease.

Adalimumab is a human monoclonal antibody against tumour necrosis factor-alpha that has been associated with acute lung toxicity, mainly in patients with rheumatoid arthritis. Descriptions of similar patterns of lung injury in patients treated with adalimumab for inflammatory bowel disease are emerging in the literature. A case involving a 45-year-old man with Crohn's disease who developed a nonbronchiolitis inflammatory nodular pattern of lung injury after starting adalimumab is reported.

Metal ion selectivity and substrate inhibition in the metal ion chelation catalyzed by human ferrochelatase.

Protoporphyrin IX ferrochelatase (EC 4.99.1.

Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction.

Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis.

Gun4 has been implicated in a developmental signaling pathway between the chloroplast and the nucleus involving magnesium protoporphyrin IX (MgP(IX)), the first dedicated intermediate in the chlorophyll biosynthetic pathway. Here we present the crystal structure of Thermosynechococcus elongatus Gun4 at 1.5 A, describe the binding affinities of Gun4 for substrate and product porphyrin molecules, and identify a likely (Mg)P(IX) binding site on the protein.

Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803.

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1).

Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.

The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change.

The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA+ ring.

The AAA(+) ATPase component of magnesium chelatase (ChlI) drives the insertion of Mg(2+) into protoporphyrin IX; this is the first step in chlorophyll biosynthesis. We describe the ATPase activity, nucleotide binding kinetics, and structural organization of the ChlI protein. A consistent reaction scheme arises from our detailed steady state description of the ATPase activity of the ChlI subunit and from transient kinetic analysis of nucleotide binding.

Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion.

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed.

Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803.

Magnesium protoporphyrin IX methyltransferase (ChlM), catalyses the methylation of magnesium protoporphyrin IX (MgP) at the C(6) propionate side chain to form magnesium protoporphyrin IX monomethylester (MgPME). Threading methods biased by sequence similarity and predicted secondary structure have been used to assign this enzyme to a particular class of S-adenosyl-L-methionine (SAM)-binding proteins. These searches suggest that ChlM contains a seven-stranded beta-sheet, common among small-molecule methyltransferases.