Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex

The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.     

Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase

In chlorophyll biosynthesis, insertion of Mg(2+) into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 A resolution to the crystallographic R-factor of 22.2 % (R(free)=24.5 %). It belongs to the chaperone-like "ATPase associated with a variety of cellular activities" (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.     

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.     

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Magnesium chelatase catalyses the insertion of Mg²⁺ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg²⁺ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg²⁺. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272?000?×?g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A₂₆₀:A₂₈₀ ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.     

EM single particle analysis of the ATP-dependent BchI complex of magnesium chelatase: an AAA+ hexamer

BchI, belonging to the AAA+ -protein family, forms the enzyme magnesium chelatase together with BchD and BchH. This enzyme catalyses the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Previous studies have indicated that BchI forms ATP-dependent complexes and it is a member of the AAA+ -protein family (ATPases associated with various cellular activities) and it was suggested based on structural homology that the BchI formed hexameric complexes. AAA+ -proteins are Mg2+ -dependent ATPases that normally form oligomeric ring complexes in the presence of ATP. Single particle analysis of fully formed ring complexes of BchI observed by negative staining EM indicate that the BchI has strong 6- and 2-fold rotational symmetries and a weaker 4-fold rotational symmetry which are reminiscent of DNA helicase. A 2D average of the fully formed BchI-ATP ring complex is presented here from images of the complex obtained from negative staining EM. Other complexes are also observed in the EM micrographs and the class averages of these are indicative of the fragility and dynamic nature of the BchI complex which has been reported and they are suggestive of partially circular complexes with six or less protomers per particle. The resolution of the average circular complex is estimated at approximately 30A and it is similar in shape and size to an atomic resolution hexameric model of BchI rendered at 30A.     

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Magnesium chelatase catalyses the insertion of Mg²⁺ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg²⁺ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg²⁺. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 × g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A₂₆₀:A₂₈₀ ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively. Received: 9 September 1996 / Accepted: 22 October 1996     

Characterization of three homologs of the large subunit of the magnesium chelatase from Chlorobaculum tepidum and interaction with the magnesium protoporphyrin IX methyltransferase

Green bacteria synthesize several types of (bacterio)chlorophylls for the assembly of functional photosynthetic reaction centers and antenna complexes. A distinctive feature of green bacteria compared with other photosynthetic microbes is that their genomes contain multiple homologs of the large subunit (BchH) of the magnesium chelatase which is a three-subunit enzyme complex (BchH, BchD, and BchI) that inserts magnesium into protoporphyrin IX as the first committed step of (bacterio)chlorophyll biosynthesis. There is speculation that the additional BchH homologs may regulate the biosynthesis of each type of chlorophyll, although the biochemical properties of the different magnesium chelatase complexes from a single species of green bacteria have not yet been compared. In this study, we investigated the activities of all three chelatase complexes from the green sulfur bacterium Chlorobaculum tepidum and interactions with the next enzyme in the pathway, magnesium protoporphyrin IX methyltransferase (BchM). Although all three chelatase complexes insert magnesium into protoporphyrin IX, the activities range by a factor of 10(5). Further, there are differences in the interactions between the BchH homologs and BchM; two of the subunits increase the methyltransferase activity by 30-60%, and the third decreases it by 30%. Expression of the chelatase complexes alone and together with BchM in Escherichia coli overproducing protoporphyrin IX suggests that the chelatase is the rate-limiting enzyme. We observed that BchM uses protoporphyrin IX without bound metal as a substrate. Our results conflict with expectations generated by previous gene inactivation studies and suggest a complex regulation of chlorophyll biosynthesis in green bacteria.     

Substrate-binding model of the chlorophyll biosynthetic magnesium chelatase BchH subunit

Photosynthetic organisms require chlorophyll and bacteriochlorophyll to harness light energy and to transform water and carbon dioxide into carbohydrates and oxygen. The biosynthesis of these pigments is initiated by magnesium chelatase, an enzyme composed of BchI, BchD, and BchH proteins, which catalyzes the insertion of Mg(2+) into protoporphyrin IX (Proto) to produce Mg-protoporphyrin IX. BchI and BchD form an ATP-dependent AAA(+) complex that transiently interacts with the Proto-binding BchH subunit, at which point Mg(2+) is chelated. In this study, controlled proteolysis, electron microscopy of negatively stained specimens, and single-particle three-dimensional reconstruction have been used to probe the structure and substrate-binding mechanism of the BchH subunit to a resolution of 25A(.) The apo structure contains three major lobe-shaped domains connected at a single point with additional densities at the tip of two lobes termed the "thumb" and "finger." With the independent reconstruction of a substrate-bound BchH complex (BchH.Proto), we observed a distinct conformational change in the thumb and finger subdomains. Prolonged proteolysis of native apo-BchH produced a stable C-terminal fragment of 45 kDa, and Proto was shown to protect the full-length polypeptide from degradation. Fitting of a truncated BchH polypeptide reconstruction identified the N- and C-terminal domains. Our results show that the N- and C-terminal domains play crucial roles in the substrate-binding mechanism.     

Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the bchI, -D, and -H Gene Products of the Green Sulfur Bacterium Chlorobium vibrioforme Expressed in Escherichia coli

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg²⁺ into protoporphyrin IX. Three genes, designated bchI, -D, and -H, from the strictly anaerobic and obligately phototrophic green sulfur bacterium Chlorobium vibrioforme show a significant level of homology to the magnesium chelatase-encoding genes bchI, -D, and -H and chlI, -D, and -H of Rhodobacter sphaeroides and Synechocystis strain PCC6803, respectively. These three genes were expressed in Escherichia coli; the subsequent purification of overproduced BchI and -H proteins on an Ni²⁺-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase of C. vibrioforme is similar to the magnesium chelatases of the distantly related bacteria R. sphaeroides and Synechocystis strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the C. vibrioforme BchI and -D proteins and the Synechocystis strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the bchI gene product, were expressed in E. coli, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in C. vibrioforme.     

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (³⁹³CX₂CX₃CX₁₄C) that was found in only six other proteobacteria (CX₂CX₃CX_11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.     

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg²⁺ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.     

Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex

Magnesium protoporphyrin chelatase catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX, which can be considered as the first committed step of (bacterio)chlorophyll synthesis. In the present work, the Mg chelatase H subunits from both Synechocystis and Rhodobacter sphaeroides were studied because of the differing requirements of these organisms for modified cyclic tetrapyrroles. Deuteroporphyrin was shown to be a substrate for Mg chelatase. Analytical HPLC gel filtration was used to show that an H-deuteroporphyrin complex can be reconstituted by incubating the magnesium chelatase H subunit with a molar excess of deuteroporphyrin and that these complexes are monomers. The binding process occurs in the absence of Mg(2+) or ATP or the I or D subunits of Mg chelatase. The emission from Trp residues in the H subunit is partly quenched when deuteroporphyrin is bound. Quantitative analysis of Trp fluorescence quenching led to determination of the K(d) values for deuteroporphyrin binding to BchH from Rb. sphaeroides and ChlH from Synechocystis, which are 1.22 +/- 0.42 microM and 0.53 +/- 0.12 microM for ChlH and BchH, respectively. In the case of ChlH, but not BchH, the K(d) increased 4-fold in the presence of MgATP(2-). Red shifts in absorbance and excitation peaks were observed in the B band of the bound porphyrin in comparison with deuteroporphyrin in solution, as well as reduced yield and red shifts of up to 8 nm in fluorescence emission. These alterations are consistent with a slightly deformed nonplanar conformation of the bound porphyrin. Mg deuteroporphyrin, the product of the Mg chelation reaction, was shown to form a complex with either ChlH or BchH; in each case the K(d) for Mg deuteroporphyrin is similar to that for deuteroporphyrin. The implications of the H-Mg protoporphyrin interaction for the next enzyme in the chlorophyll biosynthetic pathway, Mg protoporphyrin methyltransferase, are discussed.     

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 Mg²⁺ and MgATP²⁻ binding.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

Effects of Magnesium Sulfate Administration During Hypoxia on CaM Kinase IV and Protein Tyrosine Kinase Activities in the Cerebral Cortex of Newborn Piglets

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca²⁺/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg²⁺-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg²⁺-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 ± 49), Mg²⁺-Hx (317 ± 82) and Hx (574 ± 41, P < 0.05 vs. Nx and Mg²⁺-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 ± 68), Mg²⁺-Hx (455 ± 67) and Hx (922 ± 66, P < 0.05 vs. Nx and Mg²⁺-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca²⁺-flux, magnesium sulfate administration inhibits the Ca²⁺/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.     

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg²⁺ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.     

镁螯合酶的结构与功能

镁螯合酶（magnesium chelatase）是叶绿素合成过程中的关键酶,催化原卟啉IX与Mg2+螯合形成镁原卟啉IX。镁螯合酶由催化亚基H与AAA+亚基I、D组成。通过这3种亚基的协调配合,在ATP驱动下实现Mg2+与原卟啉IX的螯合,推动叶绿素的合成。在这一过程中,基因组解偶联基因4（GUN4）蛋白对其发挥重要的正调控作用。自上世纪90年代以来,镁螯合酶独特的结构及其作用机制一直吸引着研究者们的兴趣。本文结合最新的研究进展,阐述镁螯合酶的结构、酶促反应动力学及其催化机制等。另外,对于GUN4蛋白对镁螯合酶的调控也进行了概述。     

Current understanding of the function of magnesium chelatase

Despite the global significance of chlorophylls and other modified tetrapyrroles, many aspects of their biosynthetic pathways are poorly understood. A key enzyme at the branch point between the haem and chlorophyll pathways, magnesium chelatase, couples the free energy of ATP hydrolysis to the insertion of magnesium into porphyrin, a process that is likely to be mediated through protein conformational changes. Conclusions from recent structural and functional studies of individual subunits are combined to provide a mechanistic outline of the full magnesium chelatase complex. Gathering further information presents a considerable challenge, and recent steps towards this goal will be introduced.     

The AAA motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA⁺ superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA⁺ class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.     

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large‐scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe²⁺ or Mg²⁺ into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen‐regulated pufQBALMX operon encoding the reaction centre–light‐harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity and increasing cellular bacteriochlorophyll levels. FLAG‐immunoprecipitation experiments retrieve a ferrochelatase‐PufQ‐carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux toward bacteriochlorophyll production under oxygen‐limiting conditions. The co‐location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism toward bacteriochlorophyll is coordinated with the production of reaction centre and light‐harvesting polypeptides.