A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.
Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.
Kawakami
Ryushi
R
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan.
Sakuraba
Haruhiko
H
Tsuge
Hideaki
H
Goda
Shuichiro
S
Katunuma
Nobuhiko
N
Ohshima
Toshihisa
T
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000378:metabolism*; D019766:Genes, Archaeal; D008969:Molecular Sequence Data; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004396:Coloring Agents; D011994:Recombinant Proteins; D011394:Proline Oxidase