An in vitro 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 C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 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. Most of these studies have worked at a fixed saturating concentration of bicarbonate. Controlling the concentration of bicarbonate is difficult at low concentrations because of equilibration with atmospheric CO2. We describe here a simple, repeatable, and gas-tight assay system for PEPC that allows bicarbonate concentrations to be controlled above ca. 50 µM.

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. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

Kinetic Modifications of C4 PEPC Are Qualitatively Convergent, but Larger in Panicum Than in Flaveria.

C4 photosynthesis results from a set of anatomical features and biochemical components that act together to concentrate CO2 within the leaf and boost productivity. This complex trait evolved independently many times, resulting in various realizations of the phenotype, but in all C4 plants the primary fixation of atmospheric carbon is catalyzed by phosphoenolpyruvate carboxylase. Comparisons of C4 and non-C4 PEPC from a few closely related species suggested that the enzyme was modified to meet the demands of the C4 cycle. However, very few C4 groups have been investigated, hampering general conclusions. To test the hypothesis that distant C4 lineages underwent convergent biochemical changes, we compare the kinetic variation between C4 and non-C4 PEPC from a previously assessed young lineage (Flaveria, Asteraceae) with those from an older lineage found within the distantly related grass family (Panicum). Despite the evolutionary distance, the kinetic changes between the non-C4 and C4 PEPC are qualitatively similar, with a decrease in sensitivity for inhibitors, an increased specificity (k cat/K m) for bicarbonate, and a decreased specificity (k cat/K m) for PEP. The differences are more pronounced in the older lineage Panicum, which might indicate that optimization of PEPC for the C4 context increases with evolutionary time.

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. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C4 pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C4 trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate KM values similar to the non-C4 ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C4 isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.

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

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2--dependent insertion of a Mg2+ 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. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

The coproporphyrin ferrochelatase of Staphylococcus aureus: 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 Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

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/KmDIXK0.5Mgkuncat, of 30 × 10(15) m(-1) , increasing to 300 × 10(15) m(-1) with the activator protein Gun4. This is the first demonstration of the thermodynamic benefit of ATP hydrolysis in the AAA(+) family.

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. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

Characterization of the magnesium chelatase from Thermosynechococcus elongatus.

The first committed step in chlorophyll biosynthesis is catalysed by magnesium chelatase (E.C. 6.6.1.1), which uses the free energy of ATP hydrolysis to insert an Mg(2+) ion into the ring of protoporphyrin IX. We have characterized magnesium chelatase from the thermophilic cyanobacterium Thermosynechococcus elongatus. This chelatase is thermostable, with subunit melting temperatures between 55 and 63°C and optimal activity at 50°C. The T. elongatus chelatase (kcat of 0.16 µM/min) shows a Michaelis-Menten-type response to both Mg(2+) (Km of 2.3 mM) and MgATP(2-) (Km of 0.8 mM). The response to porphyrin is more complex; porphyrin inhibits at high concentrations of ChlH, but when the concentration of ChlH is comparable with the other two subunits the response is of a Michaelis-Menten type (at 0.4 µM ChlH, Km is 0.2 µM). Hybrid magnesium chelatases containing a mixture of subunits from the mesophilic Synechocystis and Thermosynechococcus enzymes are active. We generated all six possible hybrid magnesium chelatases; the hybrid chelatase containing Thermosynechococcus ChlD and Synechocystis ChlI and ChlH is not co-operative towards Mg(2+), in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.

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 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k(cat)/K(m)(app) ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k(cat) depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD(IX)) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.

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. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.

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. Kinetic analyses show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product and that it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. Gun4 therefore controls magnesium chelatase at physiologically significant Mg2+ concentrations and likely acts as a molecular switch in vivo so that in its absence magnesium chelatase is inactive. This mechanism could allow Gun4 to mediate magnesium protoporphyrin levels both for chlorophyll biosynthesis and for signaling to the nucleus.

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). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.

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. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes.

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. We provide the first demonstration of metal ion binding to a specific subunit of any of the multimeric chelatases and characterize binding of Mg(2+) to the free and MgATP(2)(-) bound forms of ChlI. Transient kinetic studies with the fluorescent substrate analogue TNP-ATP show that there are two forms of monomeric enzyme, which have distinct magnesium binding properties. Additionally, we describe the self-association properties of the subunit and provide a structural analysis of the multimeric ring formed by this enzyme in the presence of nucleotide. This single particle analysis demonstrates that this species has a 7-fold rotational symmetry, which is in marked contrast to most members of the AAA(+) family that tend to form hexamers.

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. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).