Chlorophyllide a oxidoreductase works as one of the divinyl reductases specifically involved in bacteriochlorophyll a biosynthesis

Abstract

Bacteriochlorophyll a is widely distributed among anoxygenic photosynthetic bacteria. In bacteriochlorophyll a biosynthesis, the reduction of the C8 vinyl group in 8-vinyl-chlorophyllide a is catalyzed to produce chlorophyllide a by an 8-vinyl reductase called divinyl reductase (DVR), which has been classified into two types, BciA and BciB. However, previous studies demonstrated that mutants lacking the DVR still synthesize normal bacteriochlorophyll a with the C8 ethyl group and suggested the existence of an unknown "third" DVR. Meanwhile, we recently observed that chlorophyllide a oxidoreductase (COR) of a purple bacterium happened to show the 8-vinyl reduction of 8-vinyl-chlorophyllide a in vitro. In this study, we made a double mutant lacking BciA and COR of the purple bacterium Rhodobacter sphaeroides in order to investigate whether the mutant still produces pigments with the C8 ethyl group or if COR actually works as the third DVR. The single mutant deleting BciA or COR showed production of the C8 ethyl group pigments, whereas the double mutant accumulated 8-vinyl-chlorophyllide, indicating that there was no enzyme other than BciA and COR functioning as the unknown third DVR in Rhodobacter sphaeroides (note that this bacterium has no bciB gene). Moreover, some COR genes derived from other groups of anoxygenic photosynthetic bacteria were introduced into the double mutant, and all of the complementary strains produced normal bacteriochlorophyll a. This observation indicated that COR of these bacteria performs two functions, reductions of the C8 vinyl group and the C7=C8 double bond, and that such an activity is probably conserved in the widely ranging groups.

Keywords: BciA; BciB; DVR; Evolution; Microbiology; Photosynthesis; Photosynthetic Bacteria; Photosynthetic Pigments; Purple Bacteria; Reductase.

FIGURE 1.

Proposed BChl a biosynthetic pathway, including reduction process of C8 vinyl group.

FIGURE 2.

Constructions of plasmids and bacterial mutants used in this study. Schematic maps show bciA (A) or bchX/Y/Z (B) deletion mutants of R. sphaeroides J001 strain. The neo and aadA genes confer resistance to kanamycin and streptomycin, respectively. Arrows in A and B represent the primers sphaA-F (i), sphaA-R (ii), sphaA-comf-F (iii), sphaA-comf-R (iv), sphaXYZ-F (v), sphaXYZ-R (vi), sphaXYZ-inf-F (vii), sphaXYZ-inf-R (viii), sphaXYZ-comf-F (ix), and sphaXYZ-comf-R (x). C, schematic maps showing bciB deletion mutant of R. palustris J002 strain. Arrows in C represent the primers palB-F (xi), palB-R (xii), palB-comf-F (xiii), and palB-comf-R (xiv). D–F, PCR analyses using the genomic DNA extracted from J001 (lanes 1 and 5), dA (lanes 2 and 6), dXYZ (lanes 3 and 7), dA/XYZ (lanes 4 and 8), J002 (lane 9), and dB (lane 10) strains. D, agarose gel electrophoresis (AGE) shows amplified fragments around the bciA locus using the above-mentioned sphaA-comf-F and -R primers. The PCR products from J001 and dXYZ strains (lanes 1 and 3, respectively) were 1.17 kbp, whereas the fragments from dA and dA/XYZ strains (lanes 2 and 4, respectively) were 2.50 kbp. E, the image for agarose gel electrophoresis shows amplified fragments around bchX, -Y, and -Z loci using the above-mentioned sphaXYZ-comf-F and -R primers. The PCR products from J001 and dA (lanes 5 and 6, respectively) were 4.55 kbp, whereas the fragments from dXYZ and dA/XYZ strains (lanes 7 and 8, respectively) were 2.61 kbp. F, the agarose gel electrophoresis image shows amplified fragments around the bciB locus using the above-mentioned palB-comf-F and -R primers. The PCR products from J002 and dB strains were 1.38 and 2.21 kbp, respectively. Lane M, molecular size marker (the sizes of bands are indicated at the left). Cloning of bchX, -Y, and -Z genes from C. tepidum (G) and R. castenholzii (H) is shown. Arrows in G and H represent the primers tepX-F (xv), tepX-R (xvi), tepY-F (xvii), tepY-R (xviii), tepZ-F (xix), tepZ-R (xx), castXYZ-F (xxi), castXYZ-R (xxii), cast-remove-F (xxiii), and cast-remove-R (xxiv).

FIGURE 3.

LCMS measurements of extracted pigments from the mutant cells of R. sphaeroides and R. palustris. Analyses for BChls a (A) and Chlide a derivatives (B) were monitored at 770 and 435 nm (solid lines), respectively. The dotted lines in A show mass chromatograms at m/z = 911.3 ([MH]⁺) for phytylated BChl a. i, R. sphaeroides J001 (wild type); ii, dA; iii, R. palustris J002 (wild type); iv, dB; v, dXYZ; vi, dA/XYZ; vii, standard applying Chlide a and 8V-Chlide a. Peak 1, BChl a; peak 2, geranylgeranylated BChl a; peak 3, dihydrogeranylgeranylated BChl a; peak 4, tetrahydrogeranylgeranylated BChl a; peak 5, Chlide a; peak 6, 8V-Chlide a. Peaks *5-1 and *5-2, allomers of Chlide a; peaks *6-1 and *6-2, allomers of 8V-Chlide a. C, APCI mass analysis of BChl a detected in R. sphaeroides J001 (i) and dA cells (ii). D, ESI mass spectra of Chlide a and 8V-Chlide a derivatives observed in B. Peaks *5-1, *5-2, 5, *6-1, *6-2, and 6 gave molecular ion peaks at m/z = 631.0, 631.2, 615.2, 629.2, 629.2, and 613.1, respectively, as their protonated forms ([MH]⁺). The calculated mass numbers of 631.24, 615.25, 629.23, and 613.23 are for allomer of Chlide a (and its epimer), Chlide a, allomer of 8V-Chlide a (and its epimer), and 8V-Chlide a. i, peak *5-1 found in dXYZ, allomer of Chlide a; ii, peak *5-2 found in dXYZ, allomer of Chlide a; iii, peak 5 found in dXYZ, Chlide a; iv, peak *6-1 found in dA/XYZ, allomer of 8V-Chlide a; v, peak *6-2 found in dA/XYZ, allomer of 8V-Chlide a; vi, peak 6 found in dA/XYZ, 8V-Chlide a.

FIGURE 4.

HPLC profiles detected at 435-nm absorbance (A) and selective mass chromatograms at m/z = 615.1 ([MH]⁺) for Chlide a (solid lines) and at m/z = 613.1 ([MH]⁺) for 8V-Chlide a (dashed lines) (B) of extracted pigments from the complementary strains of dA/XYZ mutant with bciA of R. sphaeroides and bciB of R. palustris. i, dA/XYZ_102 (empty vector control); ii, dA/XYZ_sphaA; iii, dA/XYZ_palB; iv, standards for Chlide a and 8V-Chlide a. Peak 5, Chlide a; peak 6, 8V-Chlide a. Peaks *5-1 and *5-2, allomers of Chlide a; peaks *6-1 and *6-2, allomers of 8V-Chlide a. The detected peaks *5-1, *5-2, 5, *6-1, *6-2, and 6 in these strains showed the same mass spectra as i, ii, iii, iv, v, and vi in Fig. 3D, respectively.

FIGURE 5.

HPLC elution profiles of extracted pigments from the complementary strains of dA/XYZ (A) and dXYZ (B) mutants with bchXYZ genes of various species. i and vii, R. sphaeroides J001 with pZJ102 plasmid; ii, dA/XYZ_102 (empty vector control); iii, dA/XYZ_sphaXYZ; iv, dA/XYZ_palXYZ; v, dA/XYZ_tepXYZ; vi, dA/XYZ_castXYZ; viii, dXYZ_102 (empty vector control); ix, dXYZ_sphaXYZ; x, dXYZ_palXYZ; xi, dXYZ_tepXYZ; xii, dXYZ_castXYZ. The elution of pigments was monitored at 770 nm. Peak 1, BChl a.