Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

The photosynthetic growth of Synechocystis sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide a (Bchlide a) in a dose-dependent manner. The growth inhibition caused by Bchlide a, however, is relieved by an increased level of exogenously added chlorophyllide a (Chlide a). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a, with inhibition constants (K_Is) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of Rhodobacter sphaeroides is inhibited competitively by Chlide a, with K_Is of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide a inhibits the metabolic conversion of exogenously added Bchlide a to bacteriochlorophyll a by an R. sphaeroides bchFNB-bchZ mutant that neither synthesizes nor metabolizes Chlide a. The metabolic inhibition by Chlide a, however, is relieved by the elevated level of Bchlide a. Thus, the chlorophyll synthase of Synechocystis sp. PCC 6803 and the bacteriochlorophyll synthase of R. sphaeroides, both of which perform ping-pong-type reactions, are inhibited by Bchlide a and Chlide a, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.The biosynthetic pathways for bacteriochlorophyll a (Bchl a) and chlorophyll a (Chl a) share the metabolic steps from protoporphyrin IX to chlorophyllide a (Chlide a) (Fig. ​(Fig.1).1). The C₂₀ moiety from geranylgeranyl pyrophosphate (GGPP) can be directly esterified to ring D of Chlide a by chlorophyll synthase (ChlG) to yield geranylgeranylated Chl a (Chl a_gg), which is subsequently reduced (at positions 6, 10, and 14 of GG) by chlorophyll reductase (ChlP) to yield phytylated Chl a (Chl a_p, but it is usually abbreviated as Chl a) (2, 7). The chlorophyll synthase of Avena sativa has a broad substrate specificity for C₂₀, and it may accept either GGPP or phytyl pyrophosphate (PPP) as the first substrate in its ping-pong-type reaction (27). Either a geranylgeranylated or phytylated enzyme esterifies the second substrate Chlide a, yielding Chl a_gg or Chl a, respectively (5, 24). Chlorophyll reductase reduces either the GG moiety of Chl a_gg or free GGPP, yielding Chl a or free PPP, respectively (25).Open in a separate windowFIG. 1.Chl a and Bchl a biosynthetic pathways (6, 7). The chemical structures of Chlide a and Bchlide a are shown. bchF codes for 3-vinyl bacteriochlorophyllide hydratase; bchXYZ for three subunits comprising COR; bchC for 3-hydroxyethyl bacteriochlorophyllide dehydrogenase; chlG and bchG for chlorophyll synthase and bacteriochlorophyll synthase, respectively; and chlP and bchP for chlorophyll reductase and bacteriochlorophyll reductase, respectively.Chlide a may be further metabolized to bacteriochlorophyllide a (Bchlide a) (Fig. ​(Fig.1).1). Chlide a reductase (COR) reduces ring B of Chlide a to form 3-vinyl bacteriochlorophyllide a, whose C-3-vinyl group on ring A is then converted into an acetyl group through the activities of hydratase (BchF) and dehydrogenase (BchC) to form Bchlide a (6). The hydratase reaction may alternatively precede that of COR (Fig. ​(Fig.1).1). Once Bchlide a is formed, its ring D is esterified with the C₂₀ geranylgeranyl moiety by bacteriochlorophyll synthase (BchG), yielding geranylgeranylated Bchl a (Bchl a_gg) (3). The C₂₀ moiety is subsequently reduced by bacteriochlorophyll reductase (BchP), yielding the phytylated Bchl a (Bchl a_p, but it is usually abbreviated as Bchl a) (1).The biosynthesis of Chl a has been regarded as a metabolism that evolved after Bchl a (33). ChlG and ChlP have been thought to emerge through the gene duplication of BchG and BchP, respectively. Recently, we found that the COR reaction, which is specific to Bchl a biosynthesis, generates superoxide at low levels of oxygen (16), and we further proposed that the degeneration of the superoxide-generating COR step may be associated with the emergence of cyanobacterium-based Chl a biosynthesis (15).The predicted sequence of ChlG of Synechocystis sp. strain PCC 6803 bears 35% identity with that of Rhodobacter sphaeroides BchG. Nonetheless, chlorophyll synthase and bacteriochlorophyll synthase exhibit a high degree of substrate specificity to distinguish their own Mg-tetrapyrrole substrates from that of the other enzyme (23, 28). We further examined whether chlorophyll synthase and bacteriochlorophyll synthase are affected by Bchlide a and Chlide a, respectively, which are structurally similar to each other. In this work, we found that the chlorophyll synthase of Synechocystis sp. PCC 6803 is competitively inhibited by Bchlide a. We further found that the bacteriochlorophyll synthase of R. sphaeroides is competitively inhibited by Chlide a. Thus, the active site of chlorophyll synthase is recognized by Bchlide a, while that of bacteriochlorophyll synthase is recognized by Chlide a. The results suggest a structural similarity between the active sites of the two enzymes.