Characterization of a Plant-like Protochlorophyllide a Divinyl Reductase in Green Sulfur Bacteria

Donald A. Bryant,Aline Gomez Maqueo Chew

doi:10.1074/jbc.m609730200

Abstract

The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur.

Highlights

The HPLC chromatogram recorded at 667 nm of the pigment extract from the CT1063::aadA mutant revealed only two Bchl cF-related peaks, indicating that methylation was only occurring at the C-121 position
The red-shifted Soret absorbance maximum is consistent with the presence of a vinyl group attached to the chlorin macrocycle, and from the absence of C-82 methylation, it can be inferred that the vinyl group must occur at the C-8 position
Both the genetic and biochemical data indicate that bciA (CT1063) and not bchJ (CT2014) encodes the C-8 vinyl reductase in C. tepidum

Summary

EXPERIMENTAL PROCEDURES

Construction of Mutants—The plating strain WT2321 of C. tepidum [16] derived from strain ATCC 49652 [17] was used in all experiments. For the HPLC and HPLC-MS analyses, cells from 1 ml of a dense culture were pelleted by centrifugation, and the pigments were extracted by sonication in acetone: methanol (7:2, v/v). The absorption spectrum of the medium was recorded as described for the cell cultures. To perform HPLC analyses of the spent growth media, the clarified medium (1 ml) was dried under vacuum and extracted with 400 ␮l of acetone:methanol (7:2, v/v). To calculate the protein:pigment ratios, cells from a dense culture (1 ml of a culture with A600 nm between 1.6 and 1.8 that had been grown at a light intensity of 90 ␮mol photons mϪ2 sϪ1) were pelleted by centrifugation. The pigments were extracted from the pellet with acetone and analyzed by HPLC and HPLC-MS as described above

RESULTS

BChl a BChl c

DISCUSSION