As protoporphyrin IX (PPn) is synthesized in a photosynthetic organism, it is chelated either with magnesium or with iron to form magnesium protoporphyrin IX (Mg-PPn) or heme, respectively. In an oxygenic photosynthetic organism, Mg-PPn is metabolized to chlorophyllide (Chlide) a, whose ring D is esterified with the C20 moiety of geranylgeranyl pyrophosphate (GGPP) by chlorophyll synthase (ChlG) (Fig. 1) to yield geranylgeranylated chlorophyll a (Chl agg) . Chlorophyll reductase (ChlP) sequentially reduces the double bonds at positions 6, 10, and 14 of the geranylgeranyl (GG) moiety to form phytylated Chl a (Chl ap or Chl a) [2,6].
Fig. 1.Reactions of ChlG and BchG. Chlide a and Bchlide a are esterified with GGPP by ChlG and BchG, respectively. The differences in chemical structure between Chlide a and Bchlide a are shaded. Ring names (A, B, C, and D) and carbon numbers (1 to 8) of tetrapyrrole are designated on Chlide a.
In anoxygenic photosynthetic organisms, Chlide a is further metabolized to bacteriochlorophyllide (Bchlide) a. Bacteriochlorophyll hydratase (BchF) hydrates the C3-vinyl group of ring A of Chlide a to form 3-hydroxyethyl Chlide a. Then, Chlide a oxidoreductase (COR) reduces ring B to form 3-hydroxyethyl Bchlide a, whose C3-hydroxyethyl group is subsequently oxidized to an acetyl group by Bchlide a dehydrogenase (BchC) . Alternatively, the reaction of COR may precede that of BchF. Recently, a broad substrate specificity of BchC of Chlorobaculum tepidum  was found, proposing a new additional sequence of reactions in the order of BchF, BchC, and COR to synthesize Bchlide a. Subsequently, the ring D of Bchlide a is esterified with the C20 moiety of GGPP by bacteriochlorophyll synthase (BchG) (Fig. 1) to yield geranylgeranylated bacteriochlorophyll a (Bchl agg) . Bacteriochlorophyll reductase (BchP) sequentially reduces the double bonds at positions 6, 10, and 14 of the GG moiety to form phytylated Bchl a (Bchl ap or Bchl a) .
The biosynthesis of Bchl a has been regarded as a metabolism that existed before the emergence of the pathway to form Chl a [12,33]. ChlG and ChlP have been thought to evolve through the duplication of genes coding for BchG and BchP, respectively. Previously, we showed that COR of Rhodobacter sphaeroides, which mediates the committing step in Bchl a biosynthesis, generates superoxide radicals when the reaction proceeds in the presence of low O2 . We further proposed that the superoxide-forming COR step, possibly along with the subsequent metabolic steps leading to Bchl a, may be degenerated as O2 was evolved from oxygenic photosynthesis . Consistent with these interpretations, the expression of COR in Synechocystis sp. PCC 6803 arrested photosynthetic growth unless expression of cytosolic superoxide dismutase is elevated .
The predicted sequence of ChlG of Synechocystis sp. PCC 6803 is considerably similar (35% identity) to that of R. sphaeroides BchG, but each enzyme has a high level of substrate specificity to distinguish its own substrate from the other [24,28]. We previously showed that ChlG of Synechocystis sp. PCC 6803 is competitively inhibited by Bchlide a, and likewise BchG of R. sphaeroides is competitively inhibited by Chlide a . Both substrates are structurally similar to each other. Thus, structural similarity would be expected between the active sites of the two enzymes.
Because the active sites of ChlG and BchG are recognized by the competitive inhibitors Bchlide a and Chlide a, respectively, we examined whether one enzyme may acquire the other enzyme activity by the mutation(s) specific to the protein. The gene chlG of Synechocystis sp. PCC 6803 was cloned into a plasmid and mobilized into the R. sphaeroides BchG mutant BG1, which is photosynthetically incompetent. Then, BG1 harboring chlG (BG1-chlG) was cultured photoheterotrophically, and mutant colonies that grew under the same conditions were obtained. All the mutants were determined to have the same I44F mutation of ChlG, and the mutated enzyme ChlGI44F was found to have BchG activity. We further mutated F to I at the corresponding 28th residue of BchG, and found that BchGF28I has ChlG activity. Thus, one synthase acquired the other enzyme activity through the substitution of the single amino acid into the residue found at the correspondi ng siteof the other enzyme, implying the importance of the residues I44 and F28 of ChlG and BchG, respectively, for substrate specificity.
Materials and Methods
Bacterial Strains and Growth Conditions
R. sphaeroides 2.4.1 (Table 1) was grown aerobically, anaerobically (with Dimethyl sulfoxide (DMSO)) in the dark, or photoheterotrophically at 28℃ in Sistrom’s succinate-based minimal medium  as described previously . Synechocystis sp. PCC 6803 was grown at 30℃ in BG11 medium  supplemented with 10 mM D-glucose as described previously . Escherichia coli was grown at 30℃ or 37℃ in Luria-Bertani medium. Antibiotics were added to the cultures of R. sphaeroides and E. coli at concentrations as indicated previously .
Table 1.aKmr, kanamycin resistance; Tcr, tetracycline resistance; Apr, ampicillin resistance.
To mutate F28 of BchG (Fig. 2A) into I, site-directed mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) according to the protocol supplied by the manufacturer. Plasmid pRbchG (Table 1) was used as a template for PCR with forward primer (5'-CCC ATC ACC TGG ATC CCG CCG ATC TG-3': mutated sequence is underlined, unless noted otherwise) and reverse primer (5'-CAG ATC GGC GGG ATC CAG GTG ATG GG-3') to yield pRbchGF28I (Table 1).
Fig. 2.Aligment of the ChlG and BchG sequences, and photoheterotrophic growth of BG1-bchG, BG1-bchGF28I, BG1-chlG, and BG1-chlGI44F. (A) The sequence of the first predicted transmembrane domain of ChlG of Synechocystis sp. PCC 6803 was compared with that of BchG of R. sphaeroides. I44 of ChlG and F28 of BchG are shaded. Keys denoting conserved sequence (*), conservative mutations (:), and semi-conservative mutations (.) are illustrated below the enzyme sequences. (B) Cells were grown under photoheterotrophic growth conditions at 10 W/m2. WT cells containing pRK415 were included as a control. The experiments were independently repeated three times; data shown are one of three representative experiments.
Construction of Plasmids
Expression of ChlG, BchG, ChlGI44F, and BchGF28I in R. sphaeroides. A 1.1 kb XbaI-EcoRI DNA fragment of pRchlG (Table 1) was cloned into the XbaI-EcoRI sites of pRK415 (Table 1) to yield pRKchlG (Table 1). Likewise, a 1.0 kb XbaI-EcoRI DNA fragment of pRbchG (Table 1) was cloned into the XbaI-EcoRI sites of pRK415 to yield pRKbchG (Table 1). The recombinant plasmid pRKchlGI44F containing the I44F mutation of ChlG (Table 1) was constructed with a 1.1 kb XbaI-EcoRI DNA fragment containing chlGI44F, which was PCR-amplified from the genome of the suppressor mutant of BG1-chlG (Table 1). Likewise, a 1.0 kb DNA fragment containing bchGF28I of pRbchGF28I was digested with XbaI and EcoRI and cloned into the XbaI-EcoRI sites of pRK415 to generate pRKbchGF28I (Table 1).
Expression of ChlG, BchG, ChlGI44F, and BchGF28I in E. coli. Plasmids pRchlG and pRbchG (Table 1) were used for the expression of ChlG and BchG in E. coli, respectively, and plasmids pRchlGI44F and pRbchGF28I (Table 1) were used for the expression of ChlGI44F and BchGF28I in E. coli, respectively. Plasmid pRchlGI44F was constructed by cloning a 1.1 kb XbaI-EcoRI DNA fragment encompassing the chlGI44F from pRKchlGI44F into the XbaI-EcoRI sites of pRSET-A (Table 1).
Determination of Light-Harvesting Complexes
R. sphaeroides was grown anaerobically (with DMSO) in the dark, and cell-free lysates were prepared as described previously . Absorption spectra of the equivalent cell-free lysates (400 μg protein) were examined with a UV 2550-PC spectrophotometer (Shimadzu, Japan). Protein levels were determined by a modified Lowry method as described previously . The amount of B800-850 complex was calculated from the spectrophotometric profile by A849-900, using an extinction coefficient (ε) of 96 mM-1 cm-1, while the amount of B875 complex was determined by A878-820 with ε of 73 mM-1 cm-1 .
Purification and Determination of Bchl a
Bchl a was purified from R. sphaeroides as described previously [7,11]. Bchl a was collected in n-hexane and its level was determined with ε of 83.9 mM-1 cm-1 at 771 nm .
Purification of Chlide a and Bchlide a
Chlide a and Bchlide a were purified from culture supernatant of BZF1 and BG1 (Table 1), respectively, as described previously . Their levels were determined with ε of 77.1 mM-1 cm-1 at 663 nm for Chlide a  and ε of 42.1 mM-1 cm-1 at 773 nm for Bchlide a . Alternatively, the C20 moieties of Chl a and Bchl a were removed by chlorophyllase to yield Chlide a and Bchlide a, respectively, as described previously [21,24]. Chl a was purchased from Sigma-Aldrich and Bchl a was purified from R. sphaeroides as described above.
Assays of ChlG, BchG, ChlGI44F, and BchGF28I
E. coli strain BL21(DE3) (Table 1) was transformed with the recombinant plasmids pRchlG, pRbchG, pRchlGI44F, and pRbchGF28I, and each recombinant strain was cultured and harvested as described previously . Reactions were performed at 30℃ and stopped by acetone as described previously . The levels of Chl a and Bchl a were determined by HPLC.
Reverse-phase HPLC analyses were performed on a LC6-AD system (Shimadzu, Japan) equipped with a Gemini C18 column (Phenomenex, Torrance, CA, USA; particle size, 5 μm; column length × diameter, 250 mm × 4.6 mm), a RF-20A fluorescence detector, and a SPD-M20A diode array detector as described previously . The fluorescence detector was set at 405 nm for excitation and at 675 nm for emission. Chl a and Bchl a were used as standards.
ChlGI44F of Synechocystis sp. PCC 6803 Supports the Photoheterotrophic Growth of R. sphaeroides BchG Mutant BG1
ChlG of Synechocystis sp. PCC 6803 and BchG of R. sphaeroides are competitively inhibited by Bchlide a and Chlide a, respectively, and the two Mg-tetrapyrroles are structurally similar to each other . Thus, the active sites of the two enzymes are thought to have similar structures, but both enzymes exhibit a high degree of substrate specificities [17,24,28]. In this work, we tried to find out the residue(s) that confer substrate specificity, and further examined whether one enzyme can acquire the other enzyme activity by mutation(s).
R. sphaeroides BG1  does not grow photoheterotrophically. Both the growth (Fig. 2B) and spectral complex formation (Fig. 3) of BG1 under anaerobic conditions were restored to wild-type (WT) levels if pRKbchG, a recombinant pRK415 carrying R. sphaeroides bchG, was provided in trans. Separately, a DNA fragment containing chlG of Synechocystis sp. PCC 6803 was cloned into pRK415 to generate pRKchlG and it was mobilized into R. sphaeroides BG1. The BG1-harboring pRKchlG (BG1-chlG) did not grow photoheterotrophically (Fig. 2B), but colonies that grew under these conditions arose at a frequency of approximately 10-8. Approximately 20 of these suppressor mutants were randomly selected from several independent experiments. Although both trans-acting (chromosomal) and cis-acting (plasmid) spontaneous mutations were expected, all the mutants showing photoheterotrophic growth were found to have cis-acting mutations. Through DNA sequencing of the plasmids from mutants, all the plasmids were determined to have the same mutational change from isoleucine (ATT) to phenylalanine (TTT) at the 44th residue of ChlG (Fig. 2A). This residue is located on the first predicted transmembrane domain of ChlG.
Fig. 3.Absorption spectra and levels of light-harvesting complexes of BG1-bchG, BG1-bchGF28I, BG1-chlG, and BG1-chlGI44F. Cells were grown anaerobically (with DMSO) in the dark in order to minimize the occurrence of suppressor mutations rescuing the growth of BG1-chlG under photoheterotrophic conditions. Absorption spectra (A) were illustrated with the levels of B800-850 complex and B875 complex (B). WT cells containing pRK415 were included as a control. The experiments were independently repeated three times; data shown are one of three representative experiments, and the average values of light-harvesting complexes are shown with standard deviation.
ChlGI44F of Synechocystis sp. PCC 6803 Shows BchG Activity
A DNA fragment containing chlGI44F was restricted from the plasmid of suppressor mutants of BG1-chlG, and recloned into pRK415 to generate pRKchlGI44F. The recombinant plasmid supported the photoheterotrophic growth of BG1 (Fig. 2B), confirming ChlGI44F indeed as a suppressor. The BG1-harboring pRKchlGI44F (BG1-chlGI44F) grew slowly compared with WT cells (Fig. 2B). The spectral complexes of BG1-chlGI44F were examined after growth under anaerobic (with DMSO) and dark conditions (Fig.3 A). The B875 complex level of BG1-chlGI44F was approximately 20% of WT level, whereas the amount of B800-850 complex was only 10% that of WT cells (Fig. 3B). The carotenoid level was also reduced relative to WT cells (Fig. 3A). The cellular Bchl a content, which amounted to 3.7 nmol/mg protein, was approximately 15% of the WT level. The results indicate that the BchG activity of ChlGI44F was not enough to synthesize WT levels of Bchl a under the conditions examined (Fig. 3B).
ChlG and ChlGI44F of Synechocystis sp. PCC 6803 were overexpressed in E. coli and used as enzyme sources. The Km of ChlGI44F for Chlide a was not different from that of ChlG, indicating no change in the affinity for Chlide a by the mutation of I44F (Table 2). However, the Km for Bchlide a was more than 10-fold larger than for Chlide a (Table 2). Thus, although ChlGI44F has BchG activity, its affinity for Bchlide a appears to be significantly lower than for its original substrate Chlide a.
Table 2.aThe experiments were independently repeated three times; data are shown as the mean ± SD. bEnzymes were expressed as his-tagged proteins in E. coli BL21(DE3). cNot applicable.
The Km of ChlGI44F for Bchlide a was further compared with that of BchG for the same substrate. R. sphaeroides BchG was overexpressed in E. coli and used as an enzyme source. The Km of BchG for Bchlide a was approximately 10 times smaller than that of ChlGI44F (Table 2). Therefore, ChlGI44F appears to have lower affinity for Bchlide a than BchG. The results may explain the slower growth (Fig. 2B) and the reduced formation of spectral complexes (Fig. 3) of BG1-chlGI44F compared with WT cells.
BchGF28I of R. sphaeroides Shows ChlG Activity
Because ChlGI44F of Synechocystis sp. PCC 6803 showed BchG activity, it was examined whether ChlG activity could be shown by BchGF28I. The amino acid F (TTC) at the 28th residue of BchG, which corresponds to the 44th of ChlG, was substituted to I (ATC). BchGF28I was overexpressed in E. coli and used as an enzyme source. Remarkably, BchGF28I of R. sphaeroides showed ChlG activity (Table 2), but the Km of BchGF28I for Chlide a was more than 15-fold larger than that for Bchlide a (Table 2). Thus, although BchGF28I has ChlG activity, its affinity for Chlide a appears to be significantly lower than for its original substrate Bchlide a. The Km of BchGF28I for Bchlide a was not different from that of BchG, indicating no change in the affinity for Bchlide a by the mutation of F28I. In accordance with the results, no difference in photoheterotrophic growth (Fig. 2B) and the spectral complexes (Fig. 3) was observed between WT cells and BG1-containing pRKbchGF28I (BG1-bchGF28I), which is a recombinant pRK415 carrying bchGF28I (Table 1).
In summary, the mutated enzymes ChlGI44F and BchGF28I showed new activities of BchG and ChlG, respectively, but the affinities for the new substrates were significantly lower than for their original ones. Thus, I44 and F28 residues are important for the substrate specificity of ChlG and BchG, respectively.
Chlide a and Bchlide a are structurally similar to each other except for the C3 functional group (vinyl and acetyl, respectively) of ring A and the redox state of the C7-C8 bond (oxidized and reduced, respectively) of ring B (Fig. 1). These structural differences between the two substrates may be prerequisites for ChlG and BchG to esterify ring D of their substrates with the C20 moiety that had been covalently attached to the enzyme using GGPP. However, the structural differences between Chlide a and Bchlide a are ignored if they bind to the active sites of BchG and ChlG, respectively, as competitive inhibitors .
Both ChlG and BchG are predicted to transverse the cell membrane nine times . Although a conserved region ([ND]-x(3)-[DE]-x(3)-D) for the binding site of polyprenyl diphosphate (GGPP and phytyl pyrophosphate [PPP]) is found at a loop between the second and third transmembrane segments , the residue responsible for prenylation and the domains for the binding of Chlide a or Bchlide a have not yet been determined. The results in this work clearly demonstrate that I44 of ChlG and F28 of BchG, which are located on the first transmembrane domain, are important for the substrate specificity for Chlide a and Bchlide a, respectively. This interpretation was further corroborated by new activities of BchG and ChlG by ChlGI44F and BchGF28I, respectively. A detailed understanding of the mechanism by which substrates interact with the I44 of ChlG and the F28 of BchG must await the determination of each enzyme structure.
The affinity of ChlGI44F for the new substrate Bchlide a appears to be lower than that of BchG. The same is true for the affinity of BchGF28I for the new substrate Chlide a. It remains to be determined whether the mutated enzymes ChlGI44F and BchGF28I can be further developed through additional mutation(s) to show the WT-level affinity for the new substrates.
Interestingly, most cyanobacterial ChlG sequences that are available in the protein database have an I at the 44th residue, but the corresponding amino acid of BchG in most purple and green phototrophic bacteria is F at this position. Chlorobaculum tepidum, a green sulfur bacterium, contains two BchG enzymes; one has F at the site corresponding to the 28th residue of R. sphaeroides BchG, whereas the other has I at the corresponding site. The two enzymes harboring F and I residues may reflect the biosynthesis of Bchl a (approximately 3% of total Chl and Bchl) and Chl a (approximately 0.3%), respectively, in addition to the major pigment Bchl c (approximately 97%) . Bchl c is mainly esterified with C15 farnesol by Bchl c synthase (BchK)  that is paralogous to BchG. The predicted sequence of BchK illustrates approximately 50% similarity to that of BchG of R. sphaeroides as well as to that of ChlG of Synechocystis sp. PCC 6803. As observed for ChlG, BchK harbors I at the site corresponding to F28 of BchG. It remains to be determined whether BchK activity can be affected by Bchlide a.
The eukaryotic green alga Chlamydomonas reinhardtii has I at the site corresponding to the 44th residue of ChlG of Synechocystis sp. PCC 6803, whereas ChlG of plants such as Avena sativa, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana contain proline at this site. Thus, it will be interesting to determine whether this residue also confers the high levels of substrate specificity as observed for ChlG of Synechocystis sp. PCC 6803.
Taken together, I44 of ChlG and its corresponding residue F28 of BchG are important in determining substrate specificity. The results in this work further reinforce the notion that ChlG and BchG are evolutionarily related enzymes. As far as we know, this is the first report on the BchG and ChlG activities of ChlGI44F and BchGF28I, respectively.