In vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives including C132-substitutes by a BciC enzyme.

Chlorosomes in the green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, e, or f molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. In this study, in vitro C132-dealkoxycarbonylations of zinc chlorophyll a derivatives bearing a methyl-, ethyl- or propyl-esterifying group and its methyl ester analogs with additional alkyl and hydroxy groups at the C132-position were examined using the BciC enzyme. The BciC-catalyzed reaction activity for the C132-methoxycarbonylated substrate was comparable to that for the ethoxycarbonylated compound; however, depropoxycarbonylation did not proceed. The BciC enzymatic demethoxycarbonylation of zinc methyl C132-alkylated pheophorbides a was gradually suppressed with the elongation of the alkyl chain and finally became inactive for the propyl substrate. The reaction of the C132-hydroxylated substrate (allomer) was accelerated compared to that of the C132-methyl analog possessing a similar steric size, and gave the corresponding C132-oxo product via further air-oxidation. All of the abovementioned enzymatic reactions occurred for one of the C132-epimers with the same configuration as in chlorophyllide a. The above substrate specificities and product distributions indicated the stereochemistry and size of the BciC enzymatic active site (pocket).

BciC-Catalyzed C132 -Demethoxycarbonylation of Metal Pheophorbide†a Alkyl Esters.

Bacteriochlorophyll c molecules self-aggregate to form large oligomers in the core part of chlorosomes, which are the main light-harvesting antenna systems of green photosynthetic bacteria. In the biosynthetic pathway of bacteriochlorophyll c, a BciC enzyme catalyzes the removal of the C132 -methoxycarbonyl group of chlorophyllide a, which possesses a free propionate residue at the C17-position and a magnesium ion as the central metal. The in vitro C132 -demethoxycarbonylations of chlorophyll a derivatives with various alkyl propionate residues and central metals were examined by using the BciC enzyme derived from one green sulfur bacteria species, Chlorobaculum tepidum. The BciC enzymatic reactions of zinc pheophorbide a alkyl esters were gradually suppressed with an increase of the alkyl chain length in the C17-propionate residue (from methyl to pentyl esters) and finally the hexyl ester became inactive for the BciC reaction. Although not only the zinc but also nickel and copper complexes were demethoxycarbonylated by the BciC enzyme, the reactions were largely dependent on the coordination ability of the central metals: Zn>Ni>Cu. The above substrate specificity indicates that the BciC enzyme would not bind directly to the carboxy group of chlorophyllide a, but would bind to its central magnesium to form the stereospecific complex of BciC with chlorophyllide a, giving pyrochlorophyllide a, which lacks the (132 R)-methoxycarbonyl group.

Stereoselective C3-substituent modification and substrate channeling by oxidoreductase BchC in bacteriochlorophyll a biosynthesis.

We report the in vitro activity of recombinant BchC oxidoreductase involved in bacteriochlorophyll a biosynthesis. BchC of Rhodobacter capsulatus preferentially oxidizes 31 R-3-(1-hydroxyethyl)-chlorophyllide a and 31 R-3-(1-hydroxyethyl)-bacteriochlorophyllide a in the presence of NAD+ to 3-acetyl-chlorophyllide a and bacteriochlorophyllide a, respectively, leaving the unreacted 31 S-epimers. In the reverse reaction, BchC with NADH predominately produces 31 R-epimeric alcohols from the 3-acetyl-(bacterio)chlorins. BchC of Chlorobaculum tepidum demonstrates the same 31 R-selectivity, suggesting that utilization of 31 R-epimers in BchC-catalyzed reductions may be conserved across different phyla of photosynthetic bacteria. Additionally, the presence of BchC accelerates the 3-vinyl hydration by BchF hydratase of Chlorobaculum tepidum during conversion of chlorophyllide a to 3-acetyl-chlorophyllide a through 3-(1-hydroxyethyl)-chlorophyllide a, indicating that these enzymes work cooperatively to promote efficient bacteriochlorophyll a biosynthesis.

In vitro demethoxycarbonylation of various chlorophyll analogs by a BciC enzyme.

Unique light-harvesting antennas in the green sulfur bacterium Chlorobaculum tepidum, called chlorosomes, consist of self-aggregates of bacteriochlorophyll (BChl) c. In the biosynthesis of BChl c, BciC demethoxycarbonylase removes the C132-methoxycarbonyl group to facilitate the self-aggregation of BChl c. We previously reported the in vitro BciC-enzymatic reactions and discussed the function of this enzyme in the biosynthesis of BChl c. This study aims to examine the substrate specificity of BciC in detail using several semi-synthetic (bacterio)chlorophyll derivatives. The results indicate that the substrate specificity of BciC is measurably affected by structural changes on the A/B rings including the bacteriochlorin π-systems. Moreover, BciC showed its activity on a Zn-chelated chlorophyll derivative. On the contrary, BciC recognized structural modifications on the D/E rings, including porphyrin pigments, which resulted in the significant decrease in the enzymatic activity. The utilization of BciC provides mild conditions that may be useful for the in vitro preparation of various chemically (un)stable chlorophyllous pigments.

In vitro enzymatic assays of photosynthetic bacterial 3-vinyl hydratases for bacteriochlorophyll biosyntheses.

A chlorosome is a large and efficient light-harvesting antenna system found in some photosynthetic bacteria. This system comprises self-aggregates of bacteriochlorophyll (BChl) c, d, or e possessing a chiral 1-hydroxyethyl group at the 3-position, which plays a key role in the formation of the supramolecule. Biosynthesis of chlorosomal pigments involves stereoselective conversion of 3-vinyl group to 3-(1-hydroxyethyl) group facilitated by a 3-vinyl hydratase. This 3-vinyl hydration also occurs in BChl a biosynthesis, followed by oxidation that introduces an acetyl group at the 3-position. Herein, we present in vitro enzymatic assays of paralogous 3-vinyl hydratases derived from green sulfur bacteria, Chlorobaculum tepidum and Chlorobaculum limnaeum, the filamentous anoxygenic phototroph Chloroflexus aurantiacus, and the chloracidobacterium Chloracidobacterium thermophilum. All the hydratases showed hydration activities. The biosynthetic pathway of BChl a and other chlorosomal pigments is discussed considering the substrate specificity and stereoselectivity of the present hydratases.

In Vitro Assays of BciC Showing C132-Demethoxycarbonylase Activity Requisite for Biosynthesis of Chlorosomal Chlorophyll Pigments.

A BciC enzyme is related to the removal of the C13(2)-methoxycarbonyl group in biosynthesis of bacteriochlorophylls (BChls) c, d and e functioning in green sulfur bacteria, filamentous anoxygenic phototrophs and phototrophic acidobacteria. These photosynthetic bacteria have the largest and the most efficient light-harvesting antenna systems, called chlorosomes, containing unique self-aggregates of BChl c, d or e pigments, that lack the C13(2)-methoxycarbonyl group which disturbs chlorosomal self-aggregation. In this study, we characterized the BciC derived from the green sulfur bacterium Chlorobaculum tepidum, and examined the in vitro enzymatic activities of its recombinant protein. The BciC-catalyzing reactions of various substrates showed that the enzyme recognized chlorophyllide (Chlide) a and 3,8-divinyl(DV)-Chlide a as chlorin substrates to give 3-vinyl-bacteriochlorophyllide (3V-BChlide) d and DV-BChlide d, respectively. Since the BciC afforded a higher activity with Chlide a than that with DV-Chlide a and no activity with (DV-)protoChlides a (porphyrin substrates) and 3V-BChlide a (a bacteriochlorin substrate), this enzyme was effective for diverting the chlorosomal pigment biosynthetic pathway at the stage of Chlide a away from syntheses of other pigments such as BChl a and Chl a The addition of methanol to the reaction mixture did not prevent the BciC activity, and we identified this enzyme as Chlide a demethoxycarbonylase, not methylesterase.

In vitro stereospecific hydration activities of the 3-vinyl group of chlorophyll derivatives by BchF and BchV enzymes involved in bacteriochlorophyll c biosynthesis of green sulfur bacteria.

The photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum produces bacteriochlorophyll (BChl) c pigments bearing a chiral 1-hydroxyethyl group at the 3-position, which self-aggregate to construct main light-harvesting antenna complexes, chlorosomes. The secondary alcoholic hydroxy group is requisite for chlorosomal aggregation and biosynthesized by hydrating the 3-vinyl group of their precursors. Using recombinant proteins of Cba. tepidum BchF and BchV, we examined in vitro enzymatic hydration of some 3-vinyl-chlorophyll derivatives. Both the enzymes catalyzed stereoselective hydration of zinc 3-vinyl-8-ethyl-12-methyl-bacteriopheophorbide c or d to the zinc 31 R-bacteriopheophorbide c or d homolog, respectively, with a slight amount of the 31 S-epimric species. A similar R-stereoselectivity was observed in the BchF-hydration of zinc 3-vinyl-8-ethyl- and propyl-12-ethyl-bacteriopheophorbides c, while their BchV-hydration gave a relatively larger amount of the 31 S-epimers. The in vitro stereoselective hydration confirmed the in vivo production of the S-epimeric species by BchV. The enzymatic hydration for the above 8-propylated substrate proceeded more slowly than that for the 8-ethylated, and the 8-isobutylated substrate was no longer hydrated. Based on these results, biosynthetic pathways of BChl c homologs and epimers are proposed.

Stereochemical conversion of C3-vinyl group to 1-hydroxyethyl group in bacteriochlorophyll c by the hydratases BchF and BchV: adaptation of green sulfur bacteria to limited-light environments.

Photosynthetic green sulfur bacteria inhabit anaerobic environments with very low-light conditions. To adapt to such environments, these bacteria have evolved efficient light-harvesting antenna complexes called as chlorosomes, which comprise self-aggregated bacteriochlorophyll c in the model green sulfur, bacterium Chlorobaculum tepidum. The pigment possess a hydroxy group at the C3(1) position that produces a chiral center with R- or S-stereochemistry and the C3(1) -hydroxy group serves as a connecting moiety for the self-aggregation. Chlorobaculum tepidum carries the two possible homologous genes for C3-vinyl hydratase, bchF and bchV. In the present study, we constructed deletion mutants of each of these genes. Pigment analyses of the bchF-inactivated mutant, which still has BchV as a sole hydratase, showed higher ratios of S-epimeric bacteriochlorophyll c than the wild-type strain. The heightened prevalence of S-stereoisomers in the mutant was more remarkable at lower light intensities and caused a red shift of the chlorosomal Qy absorption band leading to advantages for light-energy transfer. In contrast, the bchV-mutant possessing only BchF showed a significant decrease of the S-epimers and accumulations of C3-vinyl BChl c species. As trans- criptional level of bchV was upregulated at lower light intensity, the Chlorobaculum tepidum adapted to low-light environments by control of the bchV transcription.