Chorismate pyruvate-lyase and 4-hydroxy-3-solanesylbenzoate decarboxylase are required for plastoquinone biosynthesis in the cyanobacterium Synechocystis sp. PCC6803

Abstract

Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.

Keywords: 4-Hydroxy-3-prenylbenzoate Decarboxylase; Chorismate Pyruvate-Lyase; Cyanobacteria; Isoprenoid; Mutant; Photosynthesis; Plastoquinone; Quinones; Ubiquinone.

FIGURE 1.

PQ biosynthesis in Synechocystis sp. PCC6803. The chorismate pyruvate-lyase Sll1797 (this study) catalyzes the conversion of chorismate to 4-hydroxybenzoate that is prenylated by Slr0926 (8). Slr1099 (this study) and Sll0936 (this study) are both involved in the decarboxylation of 4-hydroxy-3-solanesylbenzoate. The subsequent reactions are not elucidated. A hydroxylation and two methylation reactions give likely rise to PQ. Sll0418 (5) and some unknown methyltransferases may be involved in the methylation of 2-solanesyl-1,4-benzoquinol to PQ. 1, chorismate; 2, 4-hydroxybenzoate; 3, 4-hydroxy-3-solanesylbenzoate; 4, 2-solanesylphenol; 5, 2-solanesyl-1,4-benzoquinol; 6, 3-methyl-5-solanesyl-1,4-benzoquinol; 7, plastoquinol; R, solanesyl.

FIGURE 2.

Phylogenetic tree of putative chorismate pyruvate-lyases from different prokaryotic and eukaryotic organisms. For the phylogenetic tree, sequence alignments were constructed in MEGA 5 using ClustalW (27). The maximum likelihood tree was then generated, and bootstrap values were performed with 500 replications (28). The scale bar represents 0.5 amino acid substitutions/site. Note that only the E. coli protein UbiC was characterized with regard to its catalytic activity. All analyzed sequences possess two highly conserved amino acid residues, Arg and Glu, which are likely responsible for product coordination (29). The phylogenetic tree is based on the sequences of the following organisms: cyanobacteria: Synechocystis sp. PCC6803, NP_440671 (Sll1797); G. violaceus PCC 7421, NP_924934; Microcystis aeruginosa PCC9701, ZP_18850280; Nostoc sp. PCC7107, YP_007049400; proteobacteria: E. coli K12, NP_418463 (UbiC); Vibrio cholerae, YP_004938445; Janthinobacterium sp. CG3, WP_017875115; Neisseria meningitides, WP_002240415; Bartonella elizabethae, WP_005773162; Magnetococcus marinus MC-1, YP_864172; Desulfococcus multivorans, WP_020875463; chlorobi: Chlorobium chlorochromatii CaD3, YP_380279; PVC group: Planctomyces limnophilus, YP_003628196; firmicutes: Paenibacillus sp. HW567, WP_019915017; spirochaetes: Leptospira kirschneri, WP_016751012; chlorophyta: Ostreococcus lucimarinus CCE9901 XP_001421584; C. variabilis sp. NC64A, XP_005846790; Coccomyxa subellipsoidea C-169, XP_005644772; rhodophyta: P. purpurea, NP_053990; oomycota: Phytophtora infestans T30-4, XP_002905693; and ciliophora: Tetrahymena thermophila, XP_001011395.

FIGURE 3.

Phylogenetic relationships of the Synechocystis sp. proteins Slr1099 and Sll0936 with UbiX- and UbiD-like proteins from bacteria. The maximum likelihood tree was constructed using MEGA 5 (28). The sequence alignment was conducted in MEGA 5 with Clustal W (27). Bootstrap values were performed with 1000 replications (values shown next to branches). The scale bar indicates 0.5 amino acid substitutions per site. With the exception of the Synechocystis sp. proteins and the E. coli proteins UbiX and UbiD, all other proteins were shown to contribute to 4-hydroxybenzoate decarboxylation in vitro (30–34). The reversible multisubunit hydroxyarylic decarboxylases/phenol carboxylases of Sedimentibacter hydroxybenzoicus, Bacillus subtilis, Streptomyces sp. D7, and E. coli O157:H7 are encoded by the three clustered genes: B, C, and D. The products of the genes B and C are UbiX- and UbiD-like proteins, respectively. The Synechocystis sp. genes slr1099 and sll0936 are not clustered: S. hydroxybenzoicus, ShdB (AAY67850), ShdC (AAD50377); B. subtilis, BsdB (CAB12157), BsdC (CAB12158); E. cloacae, UbiX (CAD19476), UbiD (CAD19477); Streptomyces sp., VdcB (AAD28781), VdcC (AAD28782); E. coli, UbiX (NP_288885), UbiD (NP_418285); Synechocystis sp., Slr1099 (NP_440078), Sll0936 (NP_440197); C. pneumoniae, UbiD (NP_300323), E. coli, EcdB (NP_311620), and EcdC (NP_289287).

FIGURE 4.

Growth rates and ubiquinone contents of E. coli mutants ΔubiC and ΔubiX expressing Synechocystis sp. genes. A and B, growth rates (A) and ubiquinone contents (B) of the E. coli ΔubiC expressing the Synechocystis sp. gene sll1797 (ΔubiC + sll1797) were compared with the WT and the mutant harboring the empty vector. The cells were cultivated in M59 minimal medium supplemented with 30 mm succinate. C and D, growth rates (C) and ubiquinone contents (D) of the E. coli mutant ΔubiX containing the empty vector or expressing the genes slr1099 and sll0936 were determined for cultures grown in LB medium (A and C). All data are means of at least two independent sets of experiments with S.D. < 0.01. The error bars (B and D) represent S.D. of three independent sets of experiments. 100% ubiquinone is equivalent to ∼80 nmol/g of wet weight.

FIGURE 5.

Subcellular localization and enzyme activity of Synechocystis sp. proteins expressed in E. coli. A, GST-Sll1797, GST-Slr1099, and GST-Sll0936 were expressed in E. coli BL21AI. Proteins of the 5,000 × g supernatant (cell extract, ce), 100,000 × g supernatant (s), and 100,000 × g sediment fraction (membranes, m) were separated by SDS-PAGE and analyzed by immunoblotting with GST antibodies. B, the enzyme activities of recombinant Sll1797 and Slr1099 in subcellular fractions of E. coli mutant cells ΔubiC and ΔubiX, respectively, are shown. 100% corresponds to relative specific activities of 4.5 μmol·min⁻¹·(μg protein)⁻¹ and 1.2 pmol·min⁻¹·(mg protein)⁻¹ for Sll1797- and Slr1099-containing fractions, respectively. The error bars represent the standard deviations of six independent sets of experiments. nd, not detectable; ce, 5,000 × g supernatant; s, 100,000 × g supernatant; m, 100,000 × g sediment.

FIGURE 6.

Properties of the recombinant Synechocystis sp. chorismate pyruvate-lyase Sll1797. The dependences on pH (A), protein (B), and chorismate (C) were determined. The assays were carried out in reaction mixtures containing 50 mm Bis-Tris propane/HCl buffer.

FIGURE 7.

Properties of the recombinant Synechocystis sp. decarboxylase Slr1099. A and B, the dependences on pH (A) and protein (B) were determined in assays with 50 mm Bis-Tris propane/HCl buffer and 4-hydroxy-3-geranylgeranylbenzoate as substrate. C, Slr1099 (S-1, S-2, and S-3) catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoates with different prenyl side chain lengths. Control assays (C-1, C-2, and C-3) were conducted with protein extracts from E. coli ΔubiX mutant cells harboring the empty vector. *, origin; 1, 4-hydroxy-3-farnesylbenzoate; 2, 4-hydroxy-3-geranylgeranylbenzoate; 3, 4-hydroxy-3-octaprenylbenzoate; 4, 2-farnesyl-phenol; 5, 2-geranylgeranyl-phenol; 6, 2-octaprenyl-phenol. D, the dependence on 4-hydroxy-3-prenylbenzoate was determined with 4-hydroxy-3-farnesylbenzoate (circles) and 4-hydroxy-3-geranylgeranylbenzoate (squares).

FIGURE 8.

Expression analyses of the developed Synechocystis sp. mutant strains. Partial cDNA sequences were amplified by real time quantitative PCR with gene specific primers from total RNA of Synechocystis sp. WT and mutant strains. After 40 PCR cycles, the DNA products were separated by agarose gel electrophoresis. The expression levels were compared with the housekeeping genes lysC and trpA.

FIGURE 9.

Synechocystis sp. gene disruption mutants have severe defects in growth. A, the mutant strains and the WT were cultivated in BG11 liquid medium under standard growth conditions. The growth defect of sll1797::aph was rescued by the addition of 4-hydroxybenzoate (4-HB) to the liquid medium. The final concentration was 100 μm 4-hydroxybenzoate. Mean values of two independent sets of experiments with S.D. < 0.01 are given. B, serial dilutions of cultures from the wild type and the Synechocystis sp. mutant strains sll1797::aph, slr1099::aph, and sll0936::aph were cultivated on a BG11 agar plate under standard conditions (10⁰ corresponds to A₇₃₀ 0.1).

FIGURE 10.

The first steps of the PQ biosynthetic pathway in the cyanobacterium Synechocystis sp. in comparison to ubiquinone biosynthesis in the proteobacterium E. coli and PQ biosynthesis in the higher plant A. thaliana. The dashed arrows indicate that further hydroxylation and methylation reaction steps are required for ubiquinone and PQ biosynthesis.