Characterization of Chlorophyll a and Bacteriochlorophyll a Synthases by Heterologous Expression in Escherichia coli

Abstract

Genes coding for putative chlorophyll a synthase (chlG) from Synechocystis sp. PCC 6803 and bacteriochlorophyll a synthase (bchG) from Rhodobacter capsulatus were amplified by the polymerase chain reaction and cloned into T7 RNA polymerase-based expression plasmids. In vitro enzymatic assays indicated that heterologous expression of the chlG and bchG gene products in Escherichia coli conferred chlorophyll a and bacteriochlorophyll a synthase activity, respectively. Chlorophyll a synthase utilized chlorophyllide a, but not bacteriochlorophyllide a, as a substrate, whereas bacteriochlorophyll a synthase utilized bacteriochlorophyllide a, but not chlorophyllide a. Both enzymes were also observed to exhibit a marked preference for phytyl diphosphate over geranylgeranyl diphosphate.Cyanobacteria, algae, and plants synthesize the well characterized Mg-tetrapyrrole chlorophyll a, whereas many anoxygenic photosynthetic bacteria synthesize a related, but more reduced derivative known as bacteriochlorophyll a. Analysis of Mg-tetrapyrrole intermediates that accumulate in various mutant strains of photosynthetic bacteria and algae have given rise to our current understanding of these pathways (1). As indicated in Fig. 1, both pathways utilize common intermediates from 5-aminolevulinate via Mg-protoporphyrin IX to chlorophyllide a. At this point, these pathways diverge, with the final step of chlorophyll a synthesis accomplished by the enzyme chlorophyll synthase, which catalyzes esterification of chlorophyllide a with phytyl diphosphate in green plants and with geranylgeranyl diphosphate in greening etiolated seedlings (2, 3). Esterification is a precondition for stabilization of apoproteins and for accumulation of chlorophyll-protein complexes (4, 5). The bacteriochlorophyll a biosynthetic pathway is more complex, involving a multistep modification of the chlorophyllide a ring, giving rise to a more reduced structure known as bacteriochlorophyllide a (1, 6). The final step of the bacteriochlorophyll a pathway involves esterification of bacteriochlorophyllide a catalyzed by the enzyme bacteriochlorophyll synthase (7). Bacteriochlorophyll a in most photosynthetic bacteria is esterified with phytol, but in some species, it contains geranylgeraniol instead (2). Since the bacteriochlorophyll esterification reaction has not been previously carried out in vitro, it has not been determined whether the diphosphate derivatives of the respective alcohols are the true substrates in bacteria.Genetic studies on pigment biosynthesis in Rhodobacter capsulatus initially revealed the presence of a cluster of genes involved in bacteriochlorophyll a biosynthesis (reviewed in Refs. 1 and 8). The roles of specific open reading frames in bacteriochlorophyll synthesis were determined by the construction of a defined set of interposon insertion mutations in sequenced open reading frames (6, 7, 9). One of the disruptions constructed by Bollivar et al. (6,7) resulted in the accumulation of bacteriochlorophyllide a, indicating that orf304 (bchG) may ...

BciD Is a Radical S-Adenosyl-l-methionine (SAM) Enzyme That Completes Bacteriochlorophyllide e Biosynthesis by Oxidizing a Methyl Group into a Formyl Group at C-7

“…Specifically, the enzymes responsible for adding the esterifying alcohols to Chls and BChls show considerable specificity for the (B)Chlide substrate but usually have broader specificity for the esterifying alcohol (20,(51)(52)(53). In C. tepidum BchK is responsible for the addition of the farnesyl tail to BChl c, and ChlG and BchG add phytadienol and phytol to Chl a and BChl a, respectively (20).…”

Chlorophylls, Symmetry, Chirality, and Photosynthesis

“…Lastly, esterification with a long chain alcohol then yields bacteriochlorophyll a 10. It is not yet clear whether first geranylgeranyl pyrophosphate is attached followed by reduction to phytyl or whether the reduction step precedes the esterification [150,159].…”

“…For chlorophyll a 4 the next step is simple; it involves the esterification of the C17 propionic acid group in 48 with phytyl pyrophosphate or geranylgeranyl pyrophosphate by the Chl synthase ( Figure 21) [149,150]. The enzyme is specific for chlorophyllides and cannot convert bacteriochlorophyllides.…”

Reduction Processes in Biosynthesis of Chlorophyll Molecules: Chemical Implication of Enzymatically Regio- and Stereoselective Hydrogenations in the Late Stages of Their Biosynthetic Pathway

“…71 In vitro assays indicated that (bacterio)chlorophyllides-a were phytylated in preference to being geranylgeranylated. 72 In some purple bacterial species, geranylgeranylated bacteriochlorophyll-a 63 and tetrahydrogeranylgeranylated bacteriochlorophyll-b 73 are the final products and act as photosynthetically active pigments, while bacteriochlorophyll-g possessing a non-hydrogenated farnesyl group has been exclusively observed in heliobacteria. 49 …”