Cloned (Comment) | Organism |
---|---|
gene pebB, recombinant expression of the codon optimized gene, without signal peptide, in Escherichia coli, expression of GST-tagged wild-type and mutant enzymes in Escherichia coli | Guillardia theta |
Crystallization (Comment) | Organism |
---|---|
purified substrate-free and -bound wild-type, selenomethionine-labeled enzyme PebB, hanging drop vapor diffusion method, mixing of 0.001 ml of 7 mg/ml protein solution with or without substrate, and 0.001 ml of reservoir solution containing 0.1 M Tris, pH 8.5, 34.5% PEG 3350, and 0.15 M ammonium sulfate, 4 days, 4°C, method optimization, X-ray diffraction structure determination and analysis at 1.90 A and 1.65 A, respectively | Guillardia theta |
Protein Variants | Comment | Organism |
---|---|---|
D219N | site-directed mutagenesis, inactive mutant | Guillardia theta |
D99N | site-directed mutagenesis, inactive mutant | Guillardia theta |
R215A | site-directed mutagenesis, inactive mutant | Guillardia theta |
R215K | site-directed mutagenesis, inactive mutant | Guillardia theta |
R215L | site-directed mutagenesis, inactive mutant | Guillardia theta |
R215M | site-directed mutagenesis, inactive mutant | Guillardia theta |
R215S | site-directed mutagenesis, inactive mutant | Guillardia theta |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | in cofactor ferredoxin | Guillardia theta |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
15,16-dihydrobiliverdin + reduced ferredoxin | Guillardia theta | - |
(3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? | |
15,16-dihydrobiliverdin + reduced ferredoxin | Guillardia theta CCMP2712 | - |
(3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Guillardia theta | L1IWQ9 | the gene pebB is localized on the plastid of the cryptophyte or cryptomonad | - |
Guillardia theta CCMP2712 | L1IWQ9 | the gene pebB is localized on the plastid of the cryptophyte or cryptomonad | - |
Purification (Comment) | Organism |
---|---|
recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli by DNA removal from cell extract, ultracentrifugation at 220000 x g, glutathione affinity chromatography and cleavage of the tag through TEV protease, followed by dialysis and a second step of glutathione affinity chromatography to remove that tag, gel filtration, and ultrafiltration | Guillardia theta |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(3Z)-phycoerythrobilin + oxidized ferredoxin = 15,16-dihydrobiliverdin + reduced ferredoxin + 2 H+ | analysis of the reaction mechanism, after binding of 15,16-dihydrobiliverdin (DHBV) to the enzyme, Asp99 delivers a proton to the A-ring oxygen, forming a positively charged DHBVH+. After acceptance of an electron from ferredoxin, the A-ring pyrrole proton tautomerizes to the C2 position and is stabilized there. This is facilitated by the catalytic action of the axial water, which is activated/positioned by Asp219 and (indirectly) by Arg215. For the second protonation step, the reprotonation of the A-ring nitrogen, uptake of another electron, and a final tautomerization to yield the product PEB is assumed, flipped binding mode for PEB biosynthesis | Guillardia theta |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
15,16-dihydrobiliverdin + reduced ferredoxin | - |
Guillardia theta | (3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? | |
15,16-dihydrobiliverdin + reduced ferredoxin | DHBV | Guillardia theta | (3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? | |
15,16-dihydrobiliverdin + reduced ferredoxin | - |
Guillardia theta CCMP2712 | (3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? | |
15,16-dihydrobiliverdin + reduced ferredoxin | DHBV | Guillardia theta CCMP2712 | (3Z)-phycoerythrobilin + oxidized ferredoxin | - |
? | |
additional information | the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Two central aspartate residues Asp99 and Asp219 as essential for catalytic activity. In addition, the conserved Arg215 plays a critical role in substrate specificity, binding orientation, and active site integrity | Guillardia theta | ? | - |
- |
|
additional information | the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Two central aspartate residues Asp99 and Asp219 as essential for catalytic activity. In addition, the conserved Arg215 plays a critical role in substrate specificity, binding orientation, and active site integrity | Guillardia theta CCMP2712 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
More | the structures of PebB exhibit the typical alpha/beta/alpha-sandwich fold with a central antiparallel beta-sheet, flanked by alpha-helices. Enzyme structure-function analysis, overview | Guillardia theta |
Synonyms | Comment | Organism |
---|---|---|
bilin reductase | - |
Guillardia theta |
GtPEBB | - |
Guillardia theta |
PEB:ferredoxin oxidoreductase | - |
Guillardia theta |
PebB | - |
Guillardia theta |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
assay at | Guillardia theta |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
Ferredoxin | - |
Guillardia theta |
General Information | Comment | Organism |
---|---|---|
metabolism | phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). 15,16-Dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXalpha to 15,16-DHBV, and PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. The biosynthetic intermediate DHBV is transferred via proximity channeling to PEB:ferredoxin oxidoreductase (PebB). PebB is thus far the only FDBR member that cannot use BV as a substrate. In contrast the semireduced 15,16-DHBV is employed. Upon binding, this substrate is also protonated by the central aspartate. During the reaction, a second aspartate residue gets involved and likely serves as a proton donor for the proton coupled electron transfer to the A-ring of the tetrapyrrole molecule. This reaction catalyzed by PebB is a formal reduction of the 2,3,31,32-dien system of the A-ring of 15,16-DHBV | Guillardia theta |
additional information | structure comparisons of Synechococcus WH8020 PebA and Guillardia theta PebB, overview. The Asp-99/Asp-219 pair is structurally conserved in most FDBRs, while the corresponding residues are relevant for PebB, for PebA only the homologue of Asp99 (Asp84) is essential for catalytic activity. The homologue of Asp219 (Asp205) is not essential and is rotated out of the active site. PebB binds DHBV analogous to the binding of BV in PebA/PebS | Guillardia theta |
additional information | the structures of PebB exhibit the typical alpha/beta/alpha-sandwich fold with a central antiparallel beta-sheet, flanked by alpha-helices. The open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Two central aspartate residues Asp99 and Asp219 as essential for catalytic activity. In addition, the conserved Arg215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, it is proposed that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for Aor D-ring regiospecificity. Enzyme structure-function analysis, overview | Guillardia theta |
physiological function | phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). 15,16-Dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXalpha to 15,16-DHBV, and PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB | Guillardia theta |