Tài liệu Báo cáo khoa học: A second novel dye-linked L-proline dehydrogenase complex is present in

A second novel dye-linked L-proline dehydrogenase

complex is present in the hyperthermophilic archaeon

Pyrococcus horikoshii OT-3

Ryushi Kawakami1

, Haruhiko Sakuraba1

, Hideaki Tsuge2,3, Shuichiro Goda1

, Nobuhiko Katunuma2

and Toshihisa Ohshima1

1 Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan

2 Institute for Health Science, Tokushima Bunri University, Japan

3 The Institutes for Enzyme Research, University of Tokushima, Japan

Dye-linked dehydrogenases (dye-DHs) catalyze the oxi￾dation of various amino acids, organic acids, amines

and alcohols in the presence of artificial electron accep￾tors such as 2, 6-dichloroindophenol (DCIP) and ferri￾cyanide. Although dye-DHs show a high potential for

use as specific elements in biosensors [1], their low

stability has thus far precluded their use in practical

applications and limited our ability to obtain detailed

information about their structures and functions.

Recently, however, much attention has been paid to

the isolation and characterization of enzymes from

hyperthermophilic archaea, as these organisms repre￾sent a source of extremely stable enzymes. Indeed, we

have identified several novel dye-DHs in hyperthermo￾philic archaea, including dye-linked d-proline dehy￾drogenase [2] and dye-linked l-proline dehydrogenase

Keywords

ATP-containing dehydrogenase; dye-linked

L-proline dehydrogenase; hyperthermophilic

archaeon; Pyrococcus horikoshii

Correspondence

T. Ohshima, Department of Biological

Science and Technology, Faculty of

Engineering, The University of Tokushima,

2–1 Minamijosanjima-cho, Tokushima 770–

8506, Japan

Fax: +81 88 656 9071

Tel: +81 88 656 7518

E-mail: [email protected]

(Received 7 May 2005, revised 3 June

2005, accepted 8 June 2005)

doi:10.1111/j.1742-4658.2005.04810.x

Two distinguishable activity bands for dye-linked l-proline dehydrogenase

(PDH1 and PDH2) were detected when crude extract of the hyperthermo￾philic 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 a4b4 structure,

while PDH2 has an abcd 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 b subunit bound FAD and exhibited proline dehydrogenase activity,

while the a 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 a and b subunits.

A comparison of the amino-acid sequences showed that the ADP-binding

motif in the a subunit of PDH1 clearly differs from that in the a subunit

of PDH2. It thus appears that a second novel dye-linked l-proline dehy￾drogenase complex is produced in P. horikoshii.

Abbreviations

dye-DH, dye-linked dehydrogenase; DCIP, 2,6-dichloroindophenol; dye-L-proDH, dye-linked L-proline dehydrogenase; dye-NADHDH, dye￾linked NADH dehydrogenase.

4044 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS