The enzyme, characterized from the cyanobacteria Nostoc sp. PCC 7120 and Mastigocladus laminosus, catalyses the covalent attachment of the phycobilin chromophore phycocyanobilin to cysteine 84 of the beta subunit of the phycobiliprotein phycoerythrocyanin and its isomerization to phycoviolobilin.
The enzyme, characterized from the cyanobacteria Nostoc sp. PCC 7120 and Mastigocladus laminosus, catalyses the covalent attachment of the phycobilin chromophore phycocyanobilin to cysteine 84 of the beta subunit of the phycobiliprotein phycoerythrocyanin and its isomerization to phycoviolobilin.
substrate PecA and subunit PecE form a weak complex that is stabilized by phycocyanobilin. The first reaction step involves a conformational change and/or protonation of phycocyanobilin, and subunit PecE has a chaperone-like function on the chromoprotein
the enzyme catalyses the covalent attachment of the phycobilin chromophore phycocyanobilin to cysteine 84 of the beta subunit of the phycobiliprotein phycoerythrocyanin and its isomerization to phycoviolobilin
the enzyme catalyses the covalent attachment of the phycobilin chromophore phycocyanobilin to cysteine 84 of the beta subunit of the phycobiliprotein phycoerythrocyanin and its isomerization to phycoviolobilin
2-mercaptoethanol or thiols like such as dithiothreitol are required for the isomerization reaction of the lyase: without, only the phycocyanobilin addition product is formed, but no [phycoerythrocyanin alpha-subunit]-Cys84-phycoviolobilin. Too much 2-mercaptoethanol will cause the loss of chromophore, in a reaction requiring oxygen. When Mg2+ is used as the activator, the optimal concentration of 2-mercaptoethanol is 5 mM, with Mn2+ it is 3 mM
2-mercaptoethanol or thiols like such as dithiothreitol are required for the isomerization reaction of the lyase: without, only the phycocyanobilin addition product is formed, but no [phycoerythrocyanin alpha-subunit]-Cys84-phycoviolobilin
room temperature is recommended in the absence of Triton X-100, because the proteins tend to precipitate at 37°C. In the presence of Triton X-100 (1% v/v) the temperature can be increased without precipitation to 37°C (Mg2+ as activator), or even to 45°C (Mn2+ as activator)
room temperature is recommended in the absence of Triton X-100, because the proteins tend to precipitate at 37°C. In the presence of Triton X-100 (1% v/v) the temperature can be increased without precipitation to 37°C with Mg2+ as activator
room temperature is recommended in the absence of Triton X-100, because the proteins tend to precipitate at 37°C. In the presence of Triton X-100 (1% v/v) the temperature can be increased without precipitation to 45°C with Mn2+ as activator
insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF is deleted, are constructed. All three types of mutants grew 1.3 times slower than wild-type under limiting light conditions and show a 20% reduction in the phycocyanobilin content of whole cells relative to chlorophyll alpha. Holo-phycoerythrocyanin is missing from the phycobilisomes of all three types of mutants and the level of the phycoerythrocyanin linker polypeptide is reduced relative to the wild-type
insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF is deleted, are constructed. All three types of mutants grew 1.3 times slower than wild-type under limiting light conditions and show a 20% reduction in the phycocyanobilin content of whole cells relative to chlorophyll alpha. Holo-phycoerythrocyanin is missing from the phycobilisomes of all three types of mutants and the level of the phycoerythrocyanin linker polypeptide is reduced relative to the wild-type
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
modeling of structure of the heterodimer, using the CpcE/F structure as template, cf. EC 34.4.1.32. A H87C88 motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21. The nucleophilic addition of Cys-88 to C10 of phycocyanobilin may induce the isomerization of phycocyanobilin into phycoviolobilin
when in subunit PecE the two motifs Y29YAAWWL and D263DLL are deleted, the holoenzyme loses its activity. It is also inactivated upon deletion of a central part, residues R111 to A122
when in subunit PecE the two motifs Y29YAAWWL and D263DLL are deleted, the holoenzyme loses its activity. It is also inactivated upon deletion of a central part, residues R111 to A122
when in subunit PecE the two motifs Y29YAAWWL and D263DLL are deleted, the holoenzyme loses its activity. It is also inactivated upon deletion of a central part, residues R111 to A122
when in subunit PecF the 20 C-terminal and 56 N-terminal amino acids are truncated, the lyase-isomerase activity in combination with subunit PecE decreases to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (kcat/Km) decreases 16fold when the unique four histidine residues in PecF beginning at H53 are deleted. H121 and C122 of PecF are essential for the enzyme activity, they are part of a unique stretch extending from A104 to N125 which is absent in the beta-subunit of related but non-isomerizing lyases
when in subunit PecF the 20 C-terminal and 56 N-terminal amino acids are truncated, the lyase-isomerase activity in combination with subunit PecE decreases to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (kcat/Km) decreases 16fold when the unique four histidine residues in PecF beginning at H53 are deleted. H121 and C122 of PecF are essential for the enzyme activity, they are part of a unique stretch extending from A104 to N125 which is absent in the beta-subunit of related but non-isomerizing lyases
when in subunit PecF the 20 C-terminal and 56 N-terminal amino acids are truncated, the lyase-isomerase activity in combination with subunit PecE decreases to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (kcat/Km) decreases 16fold when the unique four histidine residues in PecF beginning at H53 are deleted. H121 and C122 of PecF are essential for the enzyme activity, they are part of a unique stretch extending from A104 to N125 which is absent in the beta-subunit of related but non-isomerizing lyases
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
denaturation in 8 M urea of the individual subunits, PecE or PecF, is largely irreversible. If they are mixed together after dialysing out the urea separately, they show only little activity
Zhao, K.H.; Deng, M.G.; Zheng, M.; Zhou, M.; Parbel, A.; Storf, M.; Meyer, M.; Strohmann, B.; Scheer, H.
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon