Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis

Abstract

The biosynthesis of (bacterio)chlorophyll pigments is among the most productive biological pathways on Earth. Photosynthesis relies on these modified tetrapyrroles for the capture of solar radiation and its conversion to chemical energy. (Bacterio)chlorophylls have an isocyclic fifth ring, the formation of which has remained enigmatic for more than 60 y. This reaction is catalyzed by two unrelated cyclase enzymes using different chemistries. The majority of anoxygenic phototrophic bacteria use BchE, an O₂-sensitive [4Fe-4S] cluster protein, whereas plants, cyanobacteria, and some phototrophic bacteria possess an O₂-dependent enzyme, the major catalytic component of which is a diiron protein, AcsF. Plant and cyanobacterial mutants in ycf54 display impaired function of the O₂-dependent enzyme, accumulating the reaction substrate. Swapping cyclases between cyanobacteria and purple phototrophic bacteria reveals three classes of the O₂-dependent enzyme. AcsF from the purple betaproteobacterium Rubrivivax (Rvi.) gelatinosus rescues the loss not only of its cyanobacterial ortholog, cycI, in Synechocystis sp. PCC 6803, but also of ycf54; conversely, coexpression of cyanobacterial cycI and ycf54 is required to complement the loss of acsF in Rvi. gelatinosus These results indicate that Ycf54 is a cyclase subunit in oxygenic phototrophs, and that different classes of the enzyme exist based on their requirement for an additional subunit. AcsF is the cyclase in Rvi. gelatinosus, whereas alphaproteobacterial cyclases require a newly discovered protein that we term BciE, encoded by a gene conserved in these organisms. These data delineate three classes of O₂-dependent cyclase in chlorophototrophic organisms from higher plants to bacteria, and their evolution is discussed herein.

Keywords: bacteriochlorophyll; chlorophyll; cyclase; photosynthesis.

Fig. 1.

Cyclization reactions involved in (B)Chl biosynthesis. Shown is isocyclic ring formation via O₂-dependent and -independent routes, catalyzed by AcsF and Ycf54 (solid arrow) and BchE (dashed arrow), respectively. Here x denotes the as-yet unassigned subunit required for the O₂-dependent reaction. International Union of Pure and Applied Chemistry numbering of the relevant macrocycle carbons is indicated, and formation of the ring E is highlighted. The oxygen sources for the O₂-dependent and -independent enzymes are molecular oxygen and water, respectively.

Fig. S1.

Amino acid sequence alignments of known AcsF proteins. Sequences are those from Rvi. gelatinosus (AcsF), Synechocystis (CycI), C. reinhardtii (CRD1), A. thaliana (CHL27), and Rba. sphaeroides (Rsp_0294; abbreviated as 0294). Conserved, highly similar, and similar residues are highlighted in black, dark gray, and light gray, respectively. The putative diiron center ligands are marked by red diamonds.

Fig. 2.

Construction and phenotypic analyses of Synechocystis cyclase mutants. (A) Diagram depicting replacement of the psbAII gene with acsF^Rg via pPD[acsF^Rg] (Upper), and construction of the fully segregated strain confirmed by colony PCR (Lower). (B and C) Inactivation of cycI (B) and ycf54 (C) genes via replacement with chloramphenicol and zeocin resistance cassettes, respectively, confirmed by colony PCR. (D) Whole-cell absorption spectra of strains grown mixotrophically under low light conditions. The peaks for Chl-containing complexes are marked with a green shadow. (E) Drop growth assays of strains on solid agar, supplemented with or lacking glucose. Photographs were taken after incubation for 12 d.

Fig. S2.

Genetic knockouts and replacements in Rvi. gelatinosus. (A) Depiction of the deletion of bchE (Left), confirmed by colony PCR (Right). (B) Depiction of deletion of acsF, and subsequent integration of foreign genes at the acsF locus, under control of the native promoter (Upper), confirmed by colony PCR (Lower). The regions subjected to genetic manipulation are depicted in proportion to the scale bar. ORFs are represented as colored filled rectangles, within which the arrow indicates the direction of transcription. Crt, carotenoid biosynthesis; RC&LHC, reaction center and light-harvesting complexes.

Fig. 3.

HPLC analysis of pigments extracted from Rvi. gelatinosus strains. Pigments were extracted from the same number of cells of each strain except for the ∆bchE strain, which had a much greater BChl a content than the other strains. (A) ∆bchE. (B) ∆bchE∆acsF. (C) ∆bchE∆acsF::cycI. (D) ∆bchE∆acsF::cycI-ycf54. (E) ∆bchE∆acsF::acsF^Rs. (Inset) Retention times and Soret/Q_y maxima of peaks were used to identify BChl a.

Fig. S3.

Deletion of rsp_6110 in Rba. sphaeroides. Diagram depicting deletion of rsp_6110 (Left), and confirmation by colony PCR (Right).

Fig. 4.

HPLC analysis of pigments extracted from Rba. sphaeroides strains. Pigments were extracted from strains standardized by cell number. (A) ∆bchE∆ccoP. (B) ∆bchE∆ccoP∆6110. (C) ∆bchE∆ccoP∆6110 + pBB[6110]. (Insets) Retention times and Soret/Q_y maxima of peaks were used to identify Mg- and Zn-chelated species of BChl a.

Fig. S4.

Construction and phenotypic analysis of Rvi. gelatinosus mutant expressing bciE and acsF from Rba. sphaeroides. (A) Diagram depicting integration of bciE and acsF from Rba. sphaeroides in place of the native acsF in Rvi. gelatinosus (Upper), and confirmation by colony PCR (Lower). (B) HPLC analysis of pigments extracted from Rvi. gelatinosus strains, extracted from the same number of cells of each strain except for the ∆bchE strain, which had a much greater BChl a content compared with the other strains. (Inset) Retention times and Soret/Q_y maxima of peaks were used to identify BChl a.klj.

Fig. 5.

Phylogenetic analysis of AcsF proteins. Evolutionary analysis via a phylogenetic tree was conducted in MEGA6 using the maximum likelihood method based on the JTT matrix-based model. The analysis involved 69 protein sequences. The tree with the highest log-likelihood (−17,513.1099) is shown. Numbers next to each node indicate bootstrap values (1,000 replicates) as percentages. Phyla are distinguished by color of species name. The length of each branch represents the number of amino acid substitutions per site in proportion to the scale bar at the center of the tree. The presence/absence of BciE/Ycf54 is indicated by shading over the species names: gray, no BciE or Ycf54; orange, BciE present; green, Ycf54 present. Note that orthologs of both bciE and ycf54 are not found together in the genome of any organism sequenced to date.

Fig. S5.

Current status of known components of the oxygen-dependent cyclase. AcsF^α, AcsF^Anox, and AcsF^Ox represent AcsF proteins from Alphaproteobacteria, anoxygenic phototrophs other than the Alphaproteobacteria, and oxygenic phototrophs, respectively. e⁻ denotes the electron donor to the diiron center of AcsF.