Characterization of phycoviolobilin phycoerythrocyanin-alpha 84-cystein-lyase-(isomerizing) from Mastigocladus laminosus.
Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-alpha84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-alpha-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12-16 micro m, depending on [PecA], kcat approximately 1.2 x 10-4.s-1) and the apoprotein (Km = 2.4 micro m at 14 micro m PCB, kcat = 0.8 x 10-4.s-1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.
Zhao
Kai-Hong
KH
College of Life Science and Technology Huazhong University of Science and Technology, Wuhan, Hubei, China. khzhao@163.com
Wu
Dong
D
Wang
Lu
L
Zhou
Ming
M
Storf
Max
M
Bubenzer
Claudia
C
Strohmann
Brigitte
B
Scheer
Hugo
H
D001426:Bacterial Proteins; D015925:Cryopreservation; D000458:Cyanobacteria / Q000201:enzymology*; D003902:Detergents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000302:isolation & purification; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D012965:Sodium Chloride; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D003902:Detergents; D013438:Sulfhydryl Compounds; D008345:Manganese; D012965:Sodium Chloride; D008190:Lyases; C093743:phycoerythrocyanin alpha-subunit phycobiliviolin lyase protein, bacteria; D008274:Magnesium