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

ABSTRACT: 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.

SUBMITTER: Gibson LC

PROVIDER: S-EPMC1219958 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Project description: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.

Project description:The I, D and H subunits (ChlI, ChlD and ChlH respectively) of the magnesium protoporphyrin IX chelatase from Synechocystis have been purified to homogeneity as a result of the overexpression of the encoding genes in Escherichia coli and the production of large quantities of histidine-tagged proteins. These subunits have been used in an initial investigation of the biochemical and kinetic properties of the enzyme. The availability of pure ChlI, ChlD and ChlH has allowed us to estimate the relative concentrations of the three protein components required for optimal activity, and to investigate the dependence of chelatase activity on the concentrations of MgCl2, ATP and protoporphyrin IX. It was found that, whereas ChlD and ChlH are likely to be monomeric, ChlI can aggregate in an ATP-dependent manner, changing from a dimeric to an octameric structure. Subunit titration assays suggest an optimal ratio of ChlI, ChlD and ChlH of 2:1:4 respectively. However, the dependence of chelatase activity on increasing concentrations of ChlI and ChlH with respect to ChlD suggests that these two subunits, at least in vitro, behave as substrates in their interaction with ChlD. Mg chelation could not be detected unless the Mg2+ concentration exceeded the ATP concentration, suggesting at least two requirements for Mg2+, one as a component of MgATP2-, the other as the chelated metal. The steady-state kinetic parameters were determined from continuous assays; the Km values for protoporphyrin, MgCl2 and ATP were 1.25 microM, 4.9 mM and 0.49 mM respectively. The rate dependence of Mg2+ was clearly sigmoidal with a Hill coefficient of 3, suggesting positive co-operativity. Initiating the reaction by the addition of one of the substrates in these continuous assays resulted in a significant lag period of at least 10 min before the linear production of Mg protoporphyrin. This lag was significantly decreased by preincubating ChlI and ChlD with ATP and MgCl2, and by mixing it with ChlH that had been preincubated with protoporphyrin IX, ATP and MgCl2. This suggests not only a close MgATP2--dependent interaction between ChlI and ChlD but also an interaction between ChlH and the protoporphyrin substrate that also is stimulated by ATP and MgCl2.

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

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