Direct measurement of metal ion chelation in the active site of human ferrochelatase

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.     

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.     

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.     

Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis

Magnesium chelatase (MgCh) is a heterotrimeric enzyme complex, composed of two AAA+ family subunits that can assembly into a double ring structure and a large catalytic subunit. The small AAA+ subunit has ATPase activity and can self‐oligomerize into a ring structure, while the other AAA+ subunit lacks independent ATPase activity. Previous structural studies of the ATPase motor subunit of MgCh from a bacteriochlorophyll‐synthesizing bacterium have identified a unique ATPase clade, but the model of oligomeric assembly is unclear. Here we present the hexameric structure of the MgCh ATPase motor subunit from the chlorophyll‐synthesizing cyanobacterium Synechocystis sp. PCC 6803. This structure reveals details of how the hexameric ring is assembled, and thus provides a basis for further studying the heterotrimeric complex.     

Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB

ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP gamma S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP gamma S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA(+) proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA(+) ATPases detected both six- and sevenfold particle symmetry.     

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.     

ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.     

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.     

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo.

During biosynthesis of chlorophyll, Mg(2+) is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximately 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called "ATPases associated with various cellular activities", known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg(2+) and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.     

Domain stability in the AAA+ ATPase ClpB from Escherichia coli

ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.     

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.     

Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

TorsinA is a widely expressed AAA(+) (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.     

Distribution of ATPase and ATP-to-ADP phosphate exchange activities in magnesium chelatase subunits of Chlorobium vibrioforme and Synechocystis PCC6803

Insertion of magnesium into protoporphyrin IX is a complex ATP-dependent reaction catalysed by the enzyme Mg-chelatase. Three separate proteins (Mg-chelatase subunits), designated as D, H and I, are involved in the chelation reaction. The genes encoding the Mg-chelatase subunits of the green sulfur bacterium Chlorobium vibrioforme and of the cyanobacterium Synechocystis strain PCC6803 were expressed in Escherichia coli. The recombinant proteins were purified, tested for ATPase and phosphate exchange activities, and compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis strain PCC6803 I subunit and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmol (mg protein)^–1 min^–1, respectively. The ATPase activity of the C. vibrioforme H subunit was similar to that reported for the R. sphaeroides H subunit. The Synechocystis strain PCC6803 H subunit failed to hydrolyse ATP. The I subunit of Synechocystis strain PCC6803 and C. vibrioforme catalysed a transfer of PO₄ from ATP to ADP (exchange activity) at the rate of 1.75 ± 0.15 nmol (mg protein)^–1 min^–1. This exchange rate was 300-fold lower than that reported for the R. sphaeroides I subunit. The PO₄ exchange activities were correlated with the presence of the sequence GXRGTGKSTXVRALA in the primary structure of the three I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium [rates of 41–89 pmol Mg-deuteroporphyrin (mg protein)^–1 min^–1]. Heterologous subunit combinations resulted in low or no Mg-chelatase activity. Received: 25 May 1998 / Revision received: 24 November 1998 / Accepted: 27 November 1998     

Structure of the AAA ATPase p97

p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.     

Biochemical characterization of the apicoplast-targeted AAA+ ATPase ClpB from Plasmodium falciparum

ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins.     

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.