C. N. Hunter

A Putative Mg Chelatase Subunit from Arabidopsis thaliana cv C24 (Sequence and Transcript Analysis of the Gene, Import of the Protein into Chloroplasts, and in Situ Localization of the Transcript and Protein

We have isolated and sequenced a cDNA from Arabidopsis thaliana cv C24 that encodes a putative Mg chelatase subunit. The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrhinum majus (olive) and also high similarity to bchH, a bacterial gene involved in the Mg chelatase reaction of bacteriochlorophyll biosynthesis. We suggest that this gene be called CHL H. Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, ch-42, and ferrochelatase. The CHL H transcript was observed to undergo a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily to a minimum by the end of the light period; in contrast, transcripts for ch-42 and ferrochelatase remained constant. A model is proposed in which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle. In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the ch-42 gene. The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay. Immunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg2+. In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo.

Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI-BchD complex.

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg(2+) chelatase, such as the ATPase activity of ChlI, and the formation of ChlI-ChlD-MgATP and ChlH-protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg(2+) chelatase activity, and the formation of the ChlI-ChlD-ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI-ChlD-MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg(2+) ions. The simplest explanation for this is that in an intact Mg(2+) chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI-MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

Elucidation of the preferred routes of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis

Most of the chlorophylls and bacteriochlorophylls utilized for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803 respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series of deletions of the native bch and chl genes. In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.Some bacteria can use inorganic phosphite and hypophosphite as sources of inorganic phosphorus. Here, the authors report crystal structures of the periplasmic proteins that bind these reduced phosphorus species and show that a P-H…π interaction between the ligand and binding site determines their specificity.

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.

Five Glutamic Acid Residues in the C-Terminal Domain of the ChlD Subunit Play a Major Role in Conferring Mg2+ 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.