Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

Abstract

Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The Photosynthetic organisms synthesize both chlorophyll and heme, the two major tetrapyrroles in nature. The biosynthetic pathways of these two porphyrins utilize a number of intermediates in common and the first step unique to chlorophyll production is the insertion of Mg into protoporphyrin IX (see Fig. 1). The enzyme catalyzing this insertion is known as magnesium-protoporphyrin IX chelatase and it lies at the branch point of the heme and the bacteriochlorophyll/chlorophyll biosynthetic pathways. Despite the importance of (bacterio)chlorophyll biosynthesis, there is relatively little known about the detailed enzymology and protein chemistry of this pathway, and in the case of Mg chelatase biochemical analyses have been confined to assays using intact cells of the photosynthetic bacterium Rhodobactersphaeroides (1, 2), isolated plastids (3-6), and broken plastid systems (7)(8)(9)(10)(11)(12). In these systems, ATP is absolutely required for magnesium chelatase activity (4). Furthermore, it has been demonstrated with extracts of pea (Pisum sativum) chloroplasts that two components, one soluble and the other with membrane affinity, participate in the enzymatic reaction and that there is an ATP requirement for the activation of these two components (10). Recently, the analysis of this pathway in photosynthetic bacteria has provided a way forward (for a review, see ref. 13). This approach benefits from the availability of the genes for bacteriochlorophyll biosynthesis in Rhodobacter capsulatus and R. sphaeroides, which are clustered on a small region of the genome, -45 kb long (14-17). The gene assignments have been based on the results of insertional mutagenesis, which have been correlated with the accumulation of biosynthetic intermediates, or by the measurement of enzymatic activities (15,(17)(18)(19). Positive identification of function has been lacking, but two recent publications describe the overexpression of the bchM gene from both R. sphaeroides and R. capsulatus in Escherichia coli and demonstrate that the extracts of the E. coli transformants can convert Mg-protoporphyrin IX to Mgprotoporphyrin monomethyl ester (20,21). Apart from positively identifying bchM as the gene encoding the Mgprotoporphyrin methyltransferase, this work opens up the possibility of extending this approach to other parts of the pathway. In this paper, we report the expression of the genes bchH, -I, and -D from R. sphaeroides in E. coli: extracts from these transformants, when combined in vitro, are highly active in catalyzing the chelation of Mg by protoporphyrin IX in an ATP-dependent manner. This is an important step forward since apart from identifying the role of three more bch genes-bchH, -I, and -D-it will allow the biochemistry of this reaction to be studied in deta...

Mechanism and regulation of Mg-chelatase

“…Gibson et al confirmed this assignment biochemically, by cloning these genes into expression vectors [4]. The expressed proteins were all soluble, and when combined had Mg-chelatase activity.…”

“…The major impediment was the lack of an in itro assay, which was only achieved 6 years ago [2]. Since that time our knowledge of Mg-chelatase has expanded dramatically to the extent that the enzyme has been cloned and sequenced from several photosynthetic species [3][4][5][6][7][8][9][10].…”

“…Mg-chelatase has now been confirmed to be a multicomponent enzyme in several organisms by both biochemical assay and molecular genetics [4,6,19]. While the conclusions of Lee et al [18] may initially seem invalid, more recent data has suggested that Mg-chelatase functions as a multicomponent complex [20,21].…”

“…When the H protein is expressed in host cells, it has Proto noncovalently bound [4,21]. In the purified protein, the Proto\H subunit ratio is 1 : 1 [21].…”

“…pea, Synechocystis and Rhodobacter sequences respectively [4,6,39]. One interesting feature of all the D subunits is the proline-rich region at the centre, followed by a highly charged stretch of amino acids.…”