Molecular Genetic Analysis of Terminal Steps in Bacteriochlorophyll a Biosynthesis: Characterization of a Rhodobacter capsulatus Strain That Synthesizes Geranylgeraniol-Esterified Bacteriochlorophyll a

Bollivar, David; Wang, Shaojie; Allen, James P.; Bauer, Carl E.

doi:10.1021/bi00209a006

Cited by 81 publications

(78 citation statements)

References 34 publications

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“…Detailed investigations of the properties of chlorophyll synthase became feasible after demonstration that the bchG gene of Rhodobacter capsulatus encodes bacteriochlorophyll synthase (Bollivar et al, 1994), and the G4 gene (later named ChlG gene) of Arabidopsis thaliana encodes chlorophyll synthase (Gaubier et al, 1995). The recombinant enzyme, produced by expression of the bchG from R. capsulatus in Escherichia coli, specifically accepted bacteriochlorophyllide but not Chlide, while the analogous expression of the chlG gene from Synechococcus sp.…”

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confidence: 99%

Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

Schmid¹,

Rassadina²,

Oster³

et al. 2002

Biological Chemistry

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The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The LineweaverBurk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'ping-pong' mechanism. Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after preincubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide. Approximately 10 -17% of the recombinant enzyme were pre-loaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the well-known slow phase. This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.

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Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

Schmid¹,

Rassadina²,

Oster³

et al. 2002

Biological Chemistry

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“…In Rhodobacter capsulatus, a closely related bacterium, sequencing of the entire photosynthesis gene cluster (3), followed by directed mutational analysis of selected loci (8), has identified two genes, bchG and bchP, as responsible for the phytylation of Bchlide. It was proposed that the product of the bchG gene performs an initial esterification of Bchlide with geranylgeranyl pyrophosphate (GGPP), an activated form of the C 20 alcohol, geranylgeraniol (GG), and that this alcohol moiety is then reduced sequentially to phytol by the product of bchP (Fig.…”

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confidence: 99%

Physical Mapping of bchG , orf427 , and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides : Functional Assignment of the Bacteriochlorophyll Synthetase Gene

2000

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The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248-7255, 1999). An operonal arrangement of the genes is proposed.A rigorous program of sequencing combined with mutational and biochemical analysis has revealed within the genome of the purple bacterium Rhodobacter sphaeroides the presence of a 41-kb gene cluster holding virtually all of the loci directly responsible for its photosynthetic ability ( Fig. 1) (11,12,27), including those encoding enzymes of the bacteriochlorophyll (Bchl) biosynthesis pathway. The Bchla molecules of R. sphaeroides are esterified with phytol, a C 20 isoprenoid alcohol, which constitutes 30% of the total molecular weight and also greatly influences the molecules' properties by investing them with much of their hydrophobicity. The structural data for bacterial reaction center and light-harvesting complexes (13,15,26) enable the conformations adopted by many of the phytol chains to be examined in atomic detail. In light-harvesting complex 2 (LH2), it appears that the close intertwining of the phytol tails of the B800 and B850 Bchls, along with the carotenoid chains, imparts a significant amount of stability to the complex. Indeed, it appears that the addition of the alcohol moiety, the final act of Bchl biosynthesis in this organism, is a crucial determinant of the assembly of the entire photosynthetic apparatus (references 9 and 31 and this study). The tails also play a major role in controlling the orientation of the transition dipoles of the tetrapyrrole r...

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“…Our results, using a genetic approach to alter pigment composition, show that BchlaP does substitute for BchlaGG in vivo, in both the LH1 and RC complexes. Bollivar et al (10) working in the other direction by substituting native BchlaP with BchlaGG in Rb. capsulatus, concluded that both the light-harvesting and photochemical functions of the LH1 and RC complexes were unaffected.…”

Section: Alteration Of Pigment Synthesis In a Transconjugant Strain Omentioning

confidence: 99%

“…BchlaGG is a biosynthetic precursor of BchlaP, and the product of a single gene is responsible for the three steps (Fig. 1A) necessary for reducing BchlaGG to BchlaP (10,12,13). This gene, bchP, encodes the enzyme BchlaGG reductase and resides among other photosynthesis-related genes in a photosynthesis gene cluster (12,14,15).…”

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A Reaction Center-Light-harvesting 1 Complex (RC-LH1) from a Rhodospirillum rubrum Mutant with Altered Esterifying Pigments

Qian

Addlesee

Ruban

et al. 2003

Journal of Biological Chemistry

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Introduction of the bchP gene from Rhodobacter sphaeroides encoding geranylgeranyl reductase into Rhodospirillum rubrum alters the esterification of the bacteriochlorophylls so that phytol is used instead of geranylgeraniol. The resulting transconjugant strain of Rs. rubrum grows photosynthetically, showing that phytolated Bchla can substitute for the native pigment in both the reaction center (RC) and the light-harvesting 1 (LH1) complexes. This genetic manipulation perturbs the native carotenoid biosynthetic pathway; several biosynthetic intermediates are assembled into the core complex and are capable of energy transfer to the bacteriochlorophylls. RC-LH1 complexes containing phytolated Bchla were analyzed by low temperature absorption and fluorescence spectroscopy and circular dichroism. These show that phytolated Bchls can assemble in vivo into the photosynthetic apparatus of Rs. rubrum and that the newly introduced phytol tail provokes small perturbations to the Bchls within their binding sites in the LH1 complex. The RC-LH1 core complex was purified from membranes and reconstituted into well ordered two-dimensional crystals with a p42 1 2 space group. A projection map calculated to 9 Å shows clearly that the LH1 ring from the mutant is composed of 16 subunits that surround the reaction center and that the diameter of this complex is in close agreement with that of the wild-type LH1 complex.

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Molecular Genetic Analysis of Terminal Steps in Bacteriochlorophyll a Biosynthesis: Characterization of a Rhodobacter capsulatus Strain That Synthesizes Geranylgeraniol-Esterified Bacteriochlorophyll a

Cited by 81 publications

References 34 publications

Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

Pre-Loading of Chlorophyll Synthase with Tetraprenyl Diphosphate Is an Obligatory Step in Chlorophyll Biosynthesis

Physical Mapping of bchG , orf427 , and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides : Functional Assignment of the Bacteriochlorophyll Synthetase Gene

A Reaction Center-Light-harvesting 1 Complex (RC-LH1) from a Rhodospirillum rubrum Mutant with Altered Esterifying Pigments

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