著作: [川上 竜巳]/Noguchi Chiaki/Higashi Marie/Sakuraba Haruhiko/Ohshima Toshihisa/Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes./[Applied Microbiology and Biotechnology]
(英) Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.
(英) Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (44 and types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the subunit (PDH) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHs from 44- and -type enzyme complexes, eight PDHs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The 44-type PDHs showed greater thermostability than most of the -type PDHs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the 44-type PDHs utilized ferricyanide as the most preferable electron acceptor, whereas -type PDHs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHs likely reflect whether they were from an - or 44-type complex, though the wider divergence observed within -type PDHs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHs did not reflect the complex type. Interestingly, the k cat value for free 44-type PDH from P. horikoshii was much larger than the value for the same subunit within the 44-complex. This indicates that the isolated PDH could be a useful element for an electrochemical system for detection of L-proline.
Applied Microbiology and Biotechnology([Springer-Verlag])
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