The terminal enzymes of (bacterio)chlorophyll biosynthesis

Abstract

(Bacterio)chlorophylls are modified tetrapyrroles that are used by phototrophic organisms to harvest solar energy, powering the metabolic processes that sustain most of the life on Earth. Biosynthesis of these pigments involves enzymatic modification of the side chains and oxidation state of a porphyrin precursor, modifications that differ by species and alter the absorption properties of the pigments. (Bacterio)chlorophylls are coordinated by proteins that form macromolecular assemblies to absorb light and transfer excitation energy to a special pair of redox-active (bacterio)chlorophyll molecules in the photosynthetic reaction centre. Assembly of these pigment-protein complexes is aided by an isoprenoid moiety esterified to the (bacterio)chlorin macrocycle, which anchors and stabilizes the pigments within their protein scaffolds. The reduction of the isoprenoid 'tail' and its addition to the macrocycle are the final stages in (bacterio)chlorophyll biosynthesis and are catalysed by two enzymes, geranylgeranyl reductase and (bacterio)chlorophyll synthase. These enzymes work in conjunction with photosynthetic complex assembly factors and the membrane biogenesis machinery to synchronize delivery of the pigments to the proteins that coordinate them. In this review, we summarize current understanding of the catalytic mechanism, substrate recognition and regulation of these crucial enzymes and their involvement in thylakoid biogenesis and photosystem repair in oxygenic phototrophs.

Keywords: (bacterio)chlorophyll; ChlG; ChlP; chlorophyll synthase; geranylgeranyl reductase; photosynthesis.

Figure 1.

(Bacterio)chlorophyll biosynthesis pathways in photosynthetic organisms. The first steps of (bacterio)chlorophyll ((B)Chl) biosynthesis are shared between all photosynthetic organisms and involve chelation of a magnesium ion at the centre of the porphyrin ring and formation of the characteristic ring E producing divinyl-protochlorophyllide a (DV-PChlide a). Reduction of ring D of DV-PChlide a produces divinyl-chlorophyllide a, the last common precursor in plants, algae* and cyanobacteria, purple bacteria, heliobacteria, and ‘green bacteria’ (green sulfur bacteria, filamentous anoxygenic phototrophs (FAPs) and Acidobacteria). *Note that in some algal species variants of Chl c are produced from DV-PChlide a by action of an unknown enzyme(s) and differ depending on the identity of the ring B side chains (R1 and R2). Further specific modifications that produce the various species of (B)Chl are colour-coded according to the organism(s) in which they are synthesized along with the enzymes that catalyse the reactions. Except for Chls c, Chl biosynthesis and the biosynthesis of BChls a and b terminates with the esterification and reduction of a C20 isoprenoid moiety to ring D by ChlG/BchG and ChlP/BchP, respectively. A C15 farnesyl tail is typically added to BChlides c, d, e, f and g by BchK (BChls c–f) or BchG (BChl g). Note that some reactions may occur in alternative orders, as detailed in the text.

Figure 4.

Reactivity of modified tetrapyrrole compounds with chlorophyll synthase (ChlG). Chemical modification of chlorophyllide a (grey) to produce compounds that can be esterified by ChlG are highlighted in green and those that can no longer act as a substrate in red. ChlG requires reduction of ring D but that ring B remain oxidized, chelation of a central metal ion that forms a pentacoordinate square-pyramidal conformation, and that no bulky substituents occupy side chain positions around ring E. Modification of ring A and B side chains are tolerated. Carbon atom numbering around the tetrapyrrole macrocycle is indicated in blue.

Figure 2.

Model of the fast and slow phases of chlorophyll production by ChlG and ChlP. The model is based on data from the references described in the text. (a) Exposure of etioplasts to light activates a ternary complex consisting of protochlorophyllide reductase (POR; black), protochlorophyllide (PChlide; red) and NADPH (blue). POR catalyses electron transfer from NADPH to PChlide, producing NADP⁺ (cyan) and chlorophyllide (Chlide; dark green), respectively. A high concentration of PChlide in the prolamellar bodies (PLBs) promotes replacement of Chlide with PChlide in the ternary complex; newly released Chlide is subsequently esterified to GGPP, which is already bound to ChlG (ChlG_GGPP), followed by reduction of the GG tail to phytyl by ChlP, forming chlorophyll (Chl; green). NADPH replaces NADP⁺ and the POR cycle repeats. (b) The ensuing slow phase of Chl formation becomes prominent as the PLBs disaggregate in etio-chloroplasts and prothylakoid formation increases. PChlide becomes limiting and Chlide is not immediately released from the POR ternary complex. Instead, NADP⁺ replacement by NADPH precedes release of Chlide, thus, the flux of the Chlide substrate towards ChlG/ChlP is decreased and Chl formation slows; this is further accentuated by the need for rebinding of GGPP to ChlG.

Figure 3.

Phytyl pyrophosphate biosynthesis in oxygenic phototrophs. Dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) are condensed to form geranyl pyrophosphate (GPP) and condensation of GPP with two further molecules of IPP produces farnesyl pyrophosphate (FPP) and then geranylgeranyl pyrophosphate (GGPP). A series of three consecutive reductions of GGPP by ChlP, using NADPH as a reductant, generates phytyl pyrophosphate (PPP) via the intermediates dihydro-GGPP and tetrahydro-GGPP.

Figure 5.

Computational models of (bacterio)chlorophyll synthases. (a) Overlay of ChlG from A. thaliana (At-ChlG, dark green), ChlG from Synechocystis (Syn-ChlG, light green) and BchG from Rba. sphaeroides (Rba-BchG, purple). (b) Magnified region of the three proteins showing differences in equivalent residues (P110 in At-ChlG, I44 in Syn-ChlG and F28 in Rba-BchG) that appear to be important for substrate specificity. (c) Partially surface rendered image of ChlG (yellow) to show conserved regions between all three proteins. Fully conserved residues are shown in dark blue and conservative changes in light blue. Residues with semi-conservative changes and no homology were not surface rendered. Red spheres indicate the location of the I44 residue. (d–f) Structural models (top) and pLDDT scores (bottom) are shown for At-ChlG (d), Syn-ChlG (e) and Rba-BchG (f). Residue colours correspond to the confidence thresholds set out in Tunyasuvunakool et al. [145], with high confidence (greater than 90 pLDDT) in dark blue, reasonable confidence (90–70) in cyan, low confidence (70–50) in yellow, and very low confidence (less than 50) in red. Note that the computational simulation of At-ChlG was run using the full-annotated sequence from the UniProt database but is displayed with the N-terminal chloroplast transit peptide removed.

Figure 6.

Highly conserved residues in (bacterio)chlorophyll synthases. Structural models of ChlG from A. thaliana (At-ChlG, dark green) and Synechocystis (Syn-ChlG, light green), and BchG from Rba. sphaeroides (Rba-BchG, purple), are displayed in the left-hand panels. Conserved residues of interest are rendered in ball and stick format. Oxygen, nitrogen and sulfur atoms are coloured red, blue and yellow, respectively. (a) An expanded image of the acidic NDXXDRXXDXXXXXXR motif shows close spatial residue alignment for each structure. (b) An expanded image of the C118 position. Nearby residues that may participate in hydrophobic packing but have not been identified as critical for protein function are shown in wire format. Residue numbering corresponds to the primary sequence of A. thaliana ChlG. (c) Partial sequence alignment of Rba-BchG, Syn-ChlG, At-ChlG and the A. sativa ChlG (As-ChlG); residues from the NDXXDRXXDXXXXXXR motif (depicted in panel (a)) are highlighted in yellow and the conserved cysteine (depicted in panel (b)) in orange.