A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 Ryushi Kawakami 1 , Haruhiko Sakuraba 1 , Hideaki Tsuge 2,3 , Shuichiro Goda 1 , Nobuhiko Katunuma 2 and Toshihisa Ohshima 1 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: ohshima@bio.tokushima-u.ac.jp (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 a 4 b 4 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 (dye-l-proDH) [3,4], and found these enzymes to be highly stable and to exhibit a high potential for appli- cation in amino-acid analyses. Dye-l-proDH catalyzes the oxidation of l-proline to D 1 -pyrroline-5-carboxylate in the presence of DCIP. We first identified this enzyme in the hyperthermo- philic archaeon Thermococcus profundus DSM9503 [4]. Our functional and structural analyses showed that it is a novel bifunctional amino-acid dehydrogenase that exhibits both NADH dehydrogenase and l-proline dehydrogenase activities [3]. The enzyme is comprised of four different subunits (a, b, c and d), the genes for which form an operon [3]. A similar gene cluster also has been observed in the genome of Pyrococcus horikoshii OT-3, which has been sequenced completely [5]. During the course of screening for dye-l-proDH, we detected two electrophoretically distinguishable activity bands in the crude extract of P. horikoshii OT-3, which suggests that in addition to the abcd-type of dye-l-proDH, another, as yet unknown, dye- l-proDH is produced by this organism. In the present study, we identified the gene encoding this other enzyme, expressed it in Escherichia coli, and examined the characteristics of its product. We found the enzyme to be totally different from the abcd-type in both structure and function; that is, it is comprised of two different subunits and has no NADH dehydrogenase activity. In addition, the enzyme complex contained ATP, FMN and FAD, though abcd-type dye-l-proDH contains only FAD. Here we describe the molecular and structural characteristics of this novel, ATP-con- taining amino-acid dehydrogenase. Results and Discussion Distribution of dye-L-proDH in hyperthermophilic archaea To identify organisms that produce dye-l-proDH, we screened enzymes using native-PAGE coupled with activity staining as described in the ‘Experimental procedures.’ We observed two separate activity bands with P. horikoshii and T. peptonophilus; one band with T. profundus, P. furiosus and P. abyssi; and no activity bands with T. litoralis (data not shown). These results suggest that P. horikoshii and T. peptono- philus each produce two distinguishable forms of dye- l-proDH. Because its genome has been sequenced completely [5], we chose to purify the enzymes from P. horikoshii. Purification of PDH1 and PDH2 from P. horikoshii OT-3 and identification of the encoding genes The steps used to isolate PDH1 and PDH2 from P. horikoshii OT-3 are summarized in Tables 1 and 2, respectively. We succeeded in separating the two enzymes using Butyl Toyopearl column chromatogra- phy (Fig. 1), and were able to further purify them using the additional steps listed. Gel filtration Table 1. Purification of PDH1 from P. horikoshii. Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Yield (%) Fold Crude extract 4550 65.1 0.0143 100 1.0 Ammonium sulfate fractionation 3600 58.6 0.0163 90 1.1 DEAE Toyopearl 1040 51.7 0.0497 79 3.5 Butyl Toyopearl 295 7.40 0.0251 11 1.8 Cellulofine HAp 13.3 5.90 0.444 9 31.0 UnoQ 2.63 2.47 0.939 4 65.7 Table 2. Purification of PDH2 from P. horikoshii. Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Yield (%) Fold Crude extract 4550 65.1 0.0143 100 1.0 Ammonium sulfate fractionation 3600 58.6 0.0163 90 1.1 DEAE Toyopearl 1040 51.7 0.0497 79 3.5 Butyl Toyopearl 164 14.6 0.0890 22 6.2 Cellulofine HAp 16.2 6.71 0.414 10 30.0 UnoQ 0.536 0.752 1.40 1 97.9 R. Kawakami et al. FAD, FMN and ATP-containing amino-acid dehydrogenase FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 4045 chromatography with Superose 6 showed the molecu- lar masses of PDH1 and PDH2 to be about 440 and 101 kDa, respectively. Subjecting purified PDH1 to SDS ⁄ PAGE revealed the enzyme is comprised of two different subunits (designated a1 and b1) with mole- cular masses of about 56 and 43 kDa, respectively (data not shown). After SDS ⁄ PAGE, the stained gel was scanned, and the relative ratio of the peak areas was determined to be 1.3 (a1): 1.0 (b1) using NIH image software (http://www.rsb.info.nih.gov/nih-image/). Taking into account their molecular masses, the molecular a1: b1 ratio was calculated to be about 1 : 1, which means the enzyme has a heterooctameric (a 4 b 4 ) structure. The N-terminal amino-acid sequence of the a1 subunit was determined to be MRPLDLTEKR, which corresponds to the underlined amino-acid sequence in ML MRPLDLTEKR from the putative protein encoded by the predicted open reading frame (ORF; PH1363) based on the genome analysis. This means that ATG, which was situated 7 bp down- stream from the 5¢-terminus of the predicted ORF, is the proper initial codon for the a1 gene. The N-terminal amino-acid sequence of the b1 subunit was determined to be MLPEKSEIVV, which corresponds to that of the predicted PH1364 gene product. The a1 and b1 genes were arranged in tandem (a1 –b1) and were estimated to encode proteins with molecular masses of 55 316 and 42 685 Da, respectively. On the other hand, SDS ⁄ PAGE analysis of purified PDH2 showed four bands (data not shown). The molecular masses of the a, b, c and d subunits of PDH2 (designated a2, b2, c2 and d2) were about 52, 46, 20 and 8 kDa, respectively. Although the four dif- ferent subunits together have a mass of 126 kDa, only 101 kDa has been determined by gel filtration. On the other hand, SDS ⁄ PAGE after Superose 6 chromato- graphy showed that all four subunits were present proportionally in the active fractions. The molecular ratio of the subunits of PDH2 was determined to be 1 : 1 : 1 : 1 using the same method used for PDH1. This suggests that the enzyme has a heterotetrameric (abcd) structure. The N-terminal amino-acid sequences of the a2, b2, c2 and d2 subunits were MRINEHPILD, MIGIIGGGII, SEIPNYLKYG and MKIVCRCNDV, respectively. The N-terminal amino- acid sequence of the a2 subunit corresponded to the underlined amino-acid sequence within MEIV RINEH- PILD from the putative protein encoded by the predic- ted ORF (PH1749), except for the first methionine residue. This means that GTG, which was situated 10 bp down stream from the 5¢-terminus of the predic- ted ORF, is the proper initial codon for a2. The N-ter- minal amino-acid sequences of the b2 and c2 subunits corresponded to the sequences of the predicted PH1751 and PH1750 gene products, respectively. We previously suggested the presence of a gene encoding the d2 subunit (designated PHpdhX) [3], and the amino-acid sequence of the d2 subunit corresponded completely to that of the predicted PHpdhX gene prod- uct. These four genes were arranged in tandem (a2–c2– d2–b2) and were estimated to encode proteins with molecular masses of 52 446, 18 974, 10 076 and 42 420 Da, respectively. Although the molecular mass of d2 subunit was predicted to be 10 kDa, it was deter- mined to be 8 kDa by SDS ⁄ PAGE. This might be from the low resolution of the low molecular mass protein at the used condition (15% gel). We previously reported that the amino-acid sequences of the a, c, d and b subunits of T. profundus dye-l-proDH show a high identity with those deduced from the PH1749, PH1750, PHpdhX and PH1751 genes of P. horikoshii, respect- ively [3]. Identification of the genes encoding the four subunits of PDH2 clearly demonstrates that PDH2 is similar to the T. profundus dye-l-proDH complex. In the present study, the genes encoding the PDH1 subunits were found to form an operon (a1–b1), and similar gene clusters have been observed in the genomes of P. furiosus (PF1245–PF1246) and P. abyssi (PAB1842–PAB1843). A gene cluster like that formed by the PDH2 genes (a2–c2–d2–b2) is also distributed in these organisms [3]. Together with the results of activity screening, these observations suggest that both the a 4 b 4 and abcd dye-l-proDHs are widely distri- buted within the order Thermococcales in the archaeal Fig. 1. Elution profile obtained with Butyl Toyopearl chromatogra- phy. Enzyme solution was applied on a Butyl Toyopearl column and eluted with a linear gradient of 40–0% ammonium sulfate in buffer A. The squares and circles show the activity and A 280 , respectively. FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al. 4046 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS domain, though their expression patterns differ from one another depending up the hyperthermophilic species examined and cultivation conditions used. Expression of the PDH1 gene and purification of the recombinant enzyme We initially attempted to express the PDH1 gene using the plasmid pPDH1, but no functional product could be obtained. We therefore introduced the gene into pET11a, and were then able to successfully express it and then isolate it using the steps summarized in Table 3. E. coli strain BL21 CodonPlus RIL (DE3) cells transformed with the expression plasmid pEPDH1 exhibited a high level of dye-l-proDH activity, which was not lost upon incubation at 90 °C for 20 min. The enzyme was purified to homogeneity from cell extract using heat treatment and two successive column chro- matographies:  50 mg of the purified enzyme was obtained from 2 L of E. coli culture, and the specific activity of the enzyme was about 2-times that of the native enzyme. The purified PDH1 showed the same mobility as the native enzyme on native-PAGE, and the N-terminal amino-acid sequences of the a1 and b1 subunits of the recombinant enzyme were confirmed to be identical to those of the native enzyme. Characteristics of the recombinant PDH1 Recombinant PDH1 showed a high degree of stability against both temperature and pH (Fig. 2). The enzyme showed extreme thermostability; no activity was lost during incubation at 90 °C for 120 min (Fig. 2A). Using ferricyanide as the artificial electron acceptor, the optimum temperature for activity was determined to be about 90 °C (Fig. 2B). By contrast, T. profundus dye-l-proDH becomes completely inactive within 20 min at 80 °C [4]. Thus, PDH1 is the most thermo- stable dye-l-proDH described to date. The stability under various pH conditions was examined while incu- bating the enzyme at 50 °C for 30 min. The enzyme lost no activity between pH 5.0 and 10.0 (Fig. 2C), and the optimum pH was determined to be 7.5 (Fig. 2D). Table 3. Purification of recombinant PDH1 from E. coli. Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Yield (%) Fold Crude extract 2280 111 0.0487 100 1.0 Heat treatment 400 279 0.698 251 14.3 Butyl Toyopearl 62 113 1.82 102 37.4 Superdex 200 50 105 2.10 95 43.1 Fig. 2. (A) Thermostability of PDH1. The enzyme was incubated at 90 °C, and the residual activity was measured at the indica- ted times. (B) Effect of temperature on PDH1 activity. The enzyme activity was measured at various temperatures between 50 and 95 °C. (C) pH stability of PDH1. The enzyme was incubated at 50 °C for 30 min in buffers of various pH, after which residual activity was measured. (D) Effect of pH on PDH1 activity. The enzyme activity was measured at various pHs ranging from 6 and 9. The buffers used were Mes ⁄ NaOH (pH 6.0–7.0), Hepes ⁄ NaOH (pH 7.0–7.5) and Tris ⁄ HCl (pH 7.5–9.0). R. Kawakami et al. FAD, FMN and ATP-containing amino-acid dehydrogenase FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 4047 PDH1 acted exclusively on l-proline; l-hydroxypro- line, d-proline, cis-4-hydroxyl-d-proline, l-2 pyrroli- done-5-carboxylate, pyrrole-2-carboxylate and pipecolic acid were all inert as substrates. Although based on genome sequencing the enzyme was predicted to have sarcosine oxidase activity [5], no such activity was detec- ted. The apparent K m values for l-proline and DCIP were 4 and 0.03 mm, respectively, and the reaction product of the l-proline dehydrogenation catalyzed by PDH1 was D 1 -pyrroline-5-carboxylate. These prop- erties of PDH1 are comparable to those reported for T. profundus dye-l-proDH [4] with one noteworthy exception: PDH1 lacks the dye-linked NADH dehy- drogenase (dye-NADHDH) activity possessed by the T. profundus enzyme (see below). Amino-acid sequence alignment and functional analysis of each subunit Figures 3 and 4, respectively, show the amino-acid alignment of the a and b subunits of PDH1, PDH2 and T. profundus dye-l-proDH. The amino-acid sequence of the a1 subunit of PDH1 showed 31% and 32% identi- ties with the a2 subunit of PDH2 and the a subunit of the T. profundus enzyme, respectively (Fig. 3), while the b1 subunit showed 56% and 64% sequence identities with the b2 subunit of PDH2 and the b subunit of the T. profundus enzyme, respectively (Fig. 4). The b1 sub- unit of PDH1 contained an ADP-binding motif [6], which was well conserved in the b subunit of the abcd- type enzymes (Fig. 4). In addition, expression of the b1 gene in E. coli using a pET15b ⁄ b1 system revealed that, like the b subunit of the abcd-type enzyme [3], the b1 subunit is capable of catalyzing l-proline dehydrogena- tion by itself (data not shown). This suggests that the b1 subunit of PDH1 has the same function as that des- cribed for the b subunit of T. profundus dye-l-proDH; that is, it catalyzes the first reaction of the incorporation of electrons from l-proline into the electron transfer sys- tem [3]. We previously reported that the T. profundus enzyme exhibits dye-NADHDH activity as well as l-proline dehydrogenase activity, and functional analy- sis of each subunit showed that it is the a subunit that catalyzes the dye-NADHDH reaction [3]. We attempted to detect dye-NADHDH activity using the PDH1 com- plex, but found none. In addition, when we expressed Fig. 3. Amino-acid sequence alignment of the a subunits of PDH1, PDH2 and T. pro- fundus dye- L-proDH: alpha1, a1 subunit of PDH1; alpha2, a2 subunit of PDH2; and pdhA, a subunit of T. profundus dye- L- proDH. Asterisks(*) represent conserved residues. ADP-binding motifs [6] are boxed. FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al. 4048 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS the a1 gene in E. coli using a pET11a ⁄ a1 system, the protein produced also showed no dye-NADHDH activ- ity. Within the primary structure of the a subunit of the T. profundus enzyme are two ADP-binding motifs [6] spanning residues 120–149 and 271–300 (Fig. 3) [3]. The a1 subunit of PDH1, by contrast, contained only one ADP binding motif spanning residues 108–136 (Fig. 3), which suggests the additional ADP-binding motif in the a subunit might be essential for the dye-NADHDH activity of the abcd-type enzyme. Analysis of the prosthetic groups The absorption spectrum of PDH1 showed pro- nounced peaks at 370 and 450 nm, suggesting the pres- ence of flavin compounds. We sought to identify those compounds using high performance liquid chromatography (HPLC) and detected both FAD and FMN within the PDH1 complex (Fig. 5A); moreover, we determined there to be about 4 mol of FAD and 4 mol of FMN per mol of enzyme complex. Then using a separate expression system for each subunit gene, we found that FAD binds to the b1 subunit (Fig. 5B), which suggests that the ADP-binding motif in the b1 subunit mediates FAD binding. On the other hand, FMN was not detected bound to either subunit (Fig. 5B,C). We also extracted the flavin compounds from the native enzyme isolated from P. horikoshii cells and found that levels of FAD and FMN associ- ated with the native enzyme were similar to those seen with recombinant PDH1 (Fig. 5D). Taken together, these findings suggest that the PDH1 complex contains 1 mol of FAD per mol of b1 subunit, but that the a1 and b1 subunits separately produced in E. coli cannot bind FMN. While carrying out the procedure to identify the fla- vin compounds, we observed an unexpected signal that had about a 4-min retention time on the TSKgel ODS- 80Tm column (Fig. 5A). This signal corresponds to that of the ATP standard, and when a sample was injected together with authentic ATP, an enhancement of the peak was observed. The presence of ATP was also demonstrated using an Asahipak GS-320HQ col- umn (data not shown). The ATP content was about 4 mol of ATP per mol of enzyme complex, and ATP was present in the native enzyme as well as in the separately produced a1 subunit (Fig. 5C,D), suggesting that the enzyme complex contains 1 mol of ATP per mol of a1 subunit. The T. profundus abcd-type enzyme contains 2 mol of FAD per mol of enzyme complex [3]. As mentioned above, the a subunit of the enzyme has two ADP-binding motifs, and in a previous report we showed it to also contain 1 mol of FAD per mol of a subunit [3]. In this subunit, any other prosthetic groups than FAD were not detected [3]. As the a sub- unit exhibited dye-NADHDH activity, we supposed that one ADP-binding motif mediates FAD binding and the other is responsible for the dye-NADHDH Fig. 4. Amino-acid sequence alignment of the b subunits of PDH1, PDH2 and T. pro- fundus dye- L-proDH: beta1, b1 subunit of PDH1; beta2, b2 subunit of PDH2; and pdhB, b subunit of T. profundus dye- L- proDH. Asterisks (*) represent conserved residues. ADP-binding motifs [6] are boxed. R. Kawakami et al. FAD, FMN and ATP-containing amino-acid dehydrogenase FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 4049 activity. No flavin compounds were bound to the a1 subunit of PDH1, though it did contain one ADP- binding motif. This suggests the ADP-binding motif in the a1 subunit mediates the observed ATP binding, and FMN is likely situated at the interface between the a1 and b1 subunits within the PDH1 complex. The dependence of FMN binding on the presence of both subunits could also be due to a conformational change of one of the subunits upon heterodimer formation. There have been several reports of a family of elec- tron-transfer flavoproteins that utilize both FAD and FMN as cofactors [7–14]. This family includes cyto- chrome P450 reductase [8,9], a flavoprotein subunit of bacterial sulfite reductase [10] and nitric oxide synthase [11–14]. These enzymes mainly catalyze the transfer of reducing equivalents from NADPH to a variety of electron acceptors. In addition, recent studies have shown that members of this family have similar struc- tures consisting of two domains, one that binds FMN and one that binds FAD and NADPH [8–10]. The FMN domain is homologous to flavodoxins, while the FAD and NADPH domain is homologous to that of ferredoxin reductase [8–10,15]. PDH1 shows no similarity to any of these flavoproteins. The methylo- trophic bacterium W3A1 reportedly produces an ADP-containing oxidoreductase, trimethylamine dehy- drogenase, but not an ATP-containing dehydrogenase, and the function of ADP in trimethylamine dehydro- genase remains unknown [16]. Similarly, the catalytic properties of PDH1 can be interpreted without consid- ering the function of ATP. It may be that this ATP has a stabilizing effect on the protein, or that it plays an unknown regulatory role, as has been suggested for ADP in trimethylamine dehydrogenase. To the best of our knowledge, this is the first example of an oxido- reductase complex that contains an ATP. In general, dye-DHs play important roles in the incorporation of electrons from a substrate into the electron-transfer system. In the present study, the PDH1 complex was found to contain FAD, FMN and ATP. That PDH1 is totally different from any known electron-transfer flavoprotein that utilizes both FAD and FMN as cofactors, suggests this enzyme may employ a novel electron transfer pathway from l-pro- line to the electron-transfer system. Our goal is to bet- ter understand the relationship between the structure and function of each subunit of this enzyme and the physiological functions of the unique prosthetic groups. An X-ray reflection analysis of PDH1 is now in progress. These are essential steps in an effort to achieve the practical application of dye-l-proDHs. Experimental procedures Materials DCIP and l-proline were purchased from Nacalai Tesque (Kyoto, Japan). [ 32 P]ATP[cP] and a ProbeQuant TM G-50 microcolumn were from Amersham Bioscience (Tokyo, Japan). Restriction endonucleases were from New England Biolabs (Beverly, MA, USA). E. coli strains JM109 and BL-21 CodonPlus RIL (DE3) were from Stratagene (La Jolla, CA, USA). The plasmids, pUC18, pUC19, pET11a and pET15b were from Novagen (Tokyo, Japan). All other chemicals were of reagent grade. Microorganism and cell growth P. horikoshii OT-3 strain was obtained from the Japan Collection of Microorganisms, (Saitama, Japan), and then grown at 90 °C for 18 h under anaerobic conditions. The microorganism was cultured in medium containing 5 g of tryptone, 1 g of yeast extract, 25 g of NaCl, 1 g of cysteine- HCl, 1.3 g of (NH 4 ) 2 SO 4 , 0.28 g of KH 2 PO 4 , 0.25 g of MgSO 4 Æ7H 2 O, 0.07 g of CaCl 2 Æ2H 2 O, 0.02 g of FeCl 3 Æ6H 2 O, Fig. 5. HPLC analyses of the prosthetic groups. Elution profiles of extracts of recombinant PDH1 (A), the b subunit of PDH1 (B), the a subunit of PDH1(C), native PDH1 prepared from P. horikoshii cells (D), and the standard mixture (E). FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al. 4050 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 1.8 mg of MnCl 2 Æ4H 2 O, 4.5 mg of Na 2 B 4 O 7 Æ10H 2 O, 0.22 mg of ZnSO 4 Æ7H 2 O, 0.05 mg of CuCl 2 Æ2H 2 O, 0.03 mg of Na 2 MoO 4 Æ2H 2 O, 0.03 mg of VOSO 4 Æ2H 2 O, 0.01 mg of CoSO 4 Æ7H 2 O, and 5 g of elemental sulfur per litre (pH 6.5, adjusted with 3 m NaOH). After cultivation, the cells were collected by centrifugation (10 000 g, 15 min), washed twice with 3% NaCl, suspended in buffer A (10 mm potassium phosphate, pH 7.2 containing 1 mm EDTA) and stored at )30 °C. Enzyme assay and protein determination Enzyme activity was spectrophotometrically assayed as described previously using a Shimadzu UV-1200 spectro- photometer equipped with a thermostat [3]. Protein concentrations were determined using Bradford method; bovine serum albumin served as the standard [17]. Purification of dye-L-proDHs from P. horikoshii OT-3 P. horikoshii cells (wet weight, about 40 g) obtained from 50 L of medium were suspended in 360 mL of buffer A and disrupted by ultrasonication. After centrifugation (20 000 g, 20 min) to remove any remaining intact cells and the cell debris, the supernatant was used as the crude extract. Ammonium sulfate was added to the extract to 40% saturation, after which it was stirred at 4 °C for 1 h and centrifuged again (20 000 g, 20 min), and additional ammonium sulfate was added to the resultant supernatant containing the enzyme to bring it up to 70% saturation. Then after 1 h the solution was centrifuged, and the pre- cipitant, which contained the enzyme, was suspended in buffer A and dialyzed against the same buffer. The enzyme-containing solution was then loaded onto a DEAE Toyopearl column (4.8 · 11 cm; Tosoh, Tokyo, Japan) equilibrated with buffer A. After washing the col- umn with the same buffer, the enzyme was eluted with a 1.3-L linear gradient of 0 to 0.2 m NaCl in buffer A. The active fractions were collected, and the sample was brought up to 40% saturation with ammonium sulfate and then loaded onto a Butyl Toyopearl column (3.6 · 10 cm; Tosoh) equilibrated with buffer A supplemented with 40% ammonium sulfate. After washing the column with the same buffer, the enzyme was eluted with a 1-L linear gradi- ent of 40% to 0% ammonium sulfate in buffer A. Two peaks containing dye-l-proDH activity appeared in the elu- tion profile (Fig. 1). The enzymes corresponding to the first and second peaks were designated PDH1 and PDH2, respectively. Each enzyme solution was then dialyzed against 10 mm potassium phosphate, pH 7.2. The respective PDH1 and PDH2 solutions were sepa- rately applied to Cellulofine HAp columns (2.6 · 14 cm; Seikagaku Corp., Tokyo) equilibrated with 10 mm potas- sium phosphate, pH 7.2. After washing the columns with 100 mm potassium phosphate, pH 7.2, PDH1 was eluted with 300 mm potassium phosphate, pH 7.2, while PDH2 was eluted with 150 mm potassium phosphate, pH 7.2, and the respective active fractions were pooled and dialyzed against buffer A. PDH1 and PDH2 were then further purified separately using UnoQ (Bio-Rad, Tokyo, Japan) chromatography. The respective enzyme solutions were applied to UnoQ col- umns (0.7 · 3.5 cm) that had been equilibrated with buffer A and mounted on a fast protein liquid chromatography (FPLC) system (Bio-Rad). After washing the columns with buffer A, the enzymes were eluted with a linear gradient of 0 to 0.25 m NaCl in the same buffer, after which the respective active fractions were pooled and dialyzed against buffer A. The resultant enzyme solutions were used as the purified enzyme preparations. Preparation of the P. horikoshii genomic DNA and cloning of the enzyme genes To obtain the genomic DNA containing the PDH1 and PDH2 genes, P. horikoshii cells were first cultured as des- cribed above, filtered to remove the sulfur powder and then centrifuged (10 000 g, 15 min). The cells were then washed twice with 3% NaCl, and the OT-3 genomic DNA was pre- pared by the method of Murray and Thompson [18]. To avoid any nucleotide incorporation errors we did not use the PCR method to clone the PDH1 and PDH2 genes. Instead, two oligonucleotide probes (5¢-ATGAGACC TCTAGATCTAAC-3¢ for the PDH1 gene and 5¢-TATA TTTAGGTGGAAATTGT-3¢ for the PDH2 gene) were synthesized based on the DNA sequence in the P. horikoshii genome database, after which 1.5-pmol samples were labe- led with [ c - 32 P]ATP (1.85 MBq) using T4 polynucleotide kinase (10 U), purified on a ProbeQuant TM G-50 micro- column, and used as specific probes for southern and colony hybridizations. For preparation of the PDH1 gene, the genomic DNA was digested with SphI and KpnI; for the PDH2 gene it was digested with BamHI and SphI, and the resultant fragments were separated on 0.8% agarose gels. Approxi- mately 8.0 kbp of fragments digested with SphI and KpnI and 7.5 kbp with BamHI and SphI were extracted from the gels and inserted between the SphI and KpnI sites of plasmid pUC19 and the BamHI and SphI sites of pUC18, respectively. The E. coli strain JM 109 cells were trans- formed with these recombinant plasmids and grown on an LB plate containing 50 lgÆml )1 ampicillin, 1 mm isopro- pyl-b-d-thiogalactopyranoside (IPTG) and 200 lgÆml )1 5-bromo-4-chloro-3-indolyl-b-d-galactoside. The transform- ants were then subjected to colony hybridization as previ- ously described [2], which enabled two plasmids, pPDH1 containing the PDH1 gene (insert length; 8.1 kbp) and pPDH2 containing the PDH2 gene (insert length; 7.4 kbp), to be obtained and used as templates for DNA R. Kawakami et al. FAD, FMN and ATP-containing amino-acid dehydrogenase FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 4051 sequencing. The sequencing was carried out using the dideoxynucleotide chain-termination method [19] with an automated DNA 377 A sequencer (Applied Biosystems, Tokyo, Japan). The nucleotide sequences were analyzed using genetyx-sv ⁄ rc9.0 gene analysis software (GEN- ETYX Corp., Tokyo), and were submitted to the DDBJ under the accession numbers AB196181 (for the PDH1 gene) and AB196182 (for the PDH2 gene). Expression of the PDH1 gene and purification of its product PDH1 forms a complex comprised of two different subunits with molecular masses of 56 and 43 kDa, which were desig- nated a1 and b1, respectively; the genes encoding them were designated a1 and b1, respectively. Two sets of PCR primers were prepared to construct the expression plasmid for the PDH1 gene: 5¢-AGGGATGCATATGAGACCT CTAGATCTAAC-3¢ and 5¢-AGGCCCCGGGTCACCTC CTAGCTAGAATTC-3¢ for a1; and 5¢-AGGTGATC ATATGCTTCTAGAGAAGAGTGAAATA-3¢ and 5¢-AG AGGATCCTCAGCCCATTTGGAGGGCGG-3¢ for b1. In each case, the forward primer introduced a unique NdeI restriction site that overlapped the 5¢-initiation codon, and the reverse primer introduced a unique SmaIorBamHI restriction site proximal to the 3¢-end of the termination codon. PCR was carried out using pPDH1 as the template, after which the amplified fragments were digested with NdeI and SmaI for a1 and with NdeI and BamHI for b1. For ligation to a1, the plasmid pET11a was digested with BamHI, blunted and then further digested with NdeI. For ligation to b1, the plasmid pET11a was digested with NdeI and BamHI. The a1 and b 1 gene fragments were introduced into pET11a after linearizing it with NdeI and blunted- BamHI to generate pET11a ⁄ a1 and with NdeI and BamHI to generate pET11a ⁄ b1, respectively. pET11a ⁄ a1 was then digested with ClaI, blunted and further digested with SphI. The resultant fragment containing a1 and the T7 promoter was introduced into pET11a ⁄ b1 digested with SphI and BglII (the BglII site had already been blunted) to generate the expression plasmid pEPDH1, which was then used to transform E. coli strain BL21 CodonPlus RIL (DE3) cells. The transformants were grown for 8 h at 37 ° C in SB med- ium (1.2% tryptone peptone, 2.4% yeast extract, 1.25% K 2 HPO 4 , 0.38% KH 2 PO 4 and 0.5% glycerol) containing ampicillin (100 lgÆml )1 ), after which IPTG was added to 1mm, and cultivation was continued for an additional 4 h. The cells were then collected by centrifugation (10 000 g, 20 min), suspended in 10 mm potassium phosphate, pH 7.0 containing 1 mm DTT and disrupted by sonication. After centrifugation (20 000 g, 20 min), the supernatant was col- lected and heated at 90 °C for 10 min, the precipitant was removed by centrifugation (20 000 g, 20 min), and ammo- nium sulfate was added to the supernatant to 40% satura- tion. This enzyme solution was then applied to a Butyl Toyopearl column (2.6 · 6 cm) equilibrated with 10 mm potassium phosphate, pH 7.0 containing 0.1 mm DTT (buf- fer B) supplemented with 40% ammonium sulfate. After washing the column with the same buffer, the enzyme was eluted with a 300-mL linear gradient of 40–0% ammonium sulfate in buffer B. The active fractions were pooled, con- centrated using an Amicon Ultra-15 (30 000 MWCO), and applied to a Superdex 200 gel filtration column (2.6 · 60 cm) on an FPLC system. Buffer B containing 0.2 m NaCl was used as the elution buffer and the flow rate was 2 mL Æ min )1 . The active fractions were pooled and dia- lyzed against buffer B. All buffers used in the purification were degassed before use. Subunit gene expression and product purification Separate expression systems for the a1 and b1 subunits were constructed to determine the function of each subunit. For production of the a1 subunit, E. coli strain BL21 CodonPlus RIL (DE3) cells transformed with pET11a ⁄ a1 were grown for 6 h at 37 °C in SB medium in the presence of ampicillin (100 lgÆml )1 ), after which IPTG was added to 1mm, and cultivation was continued for an additional 3 h. The cells were then collected by centrifugation (10 000 g, 20 min), suspended in 10 mm potassium phosphate, pH 7.0 containing 1 mm DTT, and disrupted by sonication. After centrifugation (20 000 g, 20 min), the supernatant was col- lected and heated at 80 °C for 10 min and centrifuged (20 000 g, 20 min) again to remove the denatured proteins. Ammonium sulfate was added to the supernatant to 20% saturation, after which the enzyme solution was applied to a Butyl Toyopearl column (2.6 · 6 cm) equilibrated with buffer B supplemented with 20% ammonium sulfate. After washing the column with the same buffer, the enzyme was eluted with a 300-mL linear gradient of 20–0% ammonium sulfate in buffer B. The active fractions were pooled and used as the purified enzyme preparation. When a pET11a ⁄ b1 expression system was used for the production of the b1 subunit, the enzyme produced was found mainly in the insoluble fraction as an inclusion body. To avoid that, we changed the expression system to pET15b ⁄ b1. The b1 gene fragment, which had been pre- pared for construction of pET11a ⁄ b1, was introduced into plasmid pET15b linearized with NdeI and BamHI to gener- ate pET15b ⁄ b1, which was then used to transform E. coli strain BL21 CodonPlus RIL (DE3) cells. The transformants were grown for 6 h at 37 °C in SB medium in the presence of ampicillin (100 lgÆml )1 ), after which IPTG was added to 1mm, and cultivation was continued for an additional 3 h. The cells were then collected by centrifugation, suspended in 10 mm Tris ⁄ HCl, pH 7.5, and disrupted by sonication. After centrifugation, imidazole and NaCl were added to the supernatant to 50 mm and 0.5 m, respectively, and the resultant solution was applied to a HiTrap nickel-charged chelating column (2.6 · 6 cm; Amersham Biosciences) FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al. 4052 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS equilibrated with 10 mm Tris ⁄ HCl, pH 7.5 containing 50 mm imidazole and 0.5 m NaCl. After washing the col- umn with the same buffer, the enzyme was eluted with 10 mm Tris ⁄ HCl, pH 7.5 containing 500 mm imidazole and 0.5 m NaCl. The active fractions were pooled, heated at 70 °C for 10 min and centrifuged to remove the precipit- ants, and then the supernatant was dialyzed against 10 mm Tris ⁄ HCl, pH 7.5. The resultant enzyme solution was used as the purified preparation. All buffers used in the purifica- tion were degassed before use. Determination of flavin and other prosthetic groups Flavin compounds and other prosthetic groups from the enzyme were extracted with 1% (w ⁄ v) perchloric acid. The solution was then centrifuged, and after neutralizing the supernatant with K 2 CO 3 , it was subjected to HPLC using TSKgel ODS-80Tm (Tosoh) and Asahipak GS-320HQ (Shodex, Tokyo) columns. An isocratic elution (10 min) with 10 mm potassium phosphate, pH 6.0 followed by a lin- ear gradient (30 min) between 0 and 70% methanol in the same solution was used for the TSKgel ODS-80Tm column. An isocratic elution with 200 mm sodium phosphate, pH 5.0 was used for the Asahipak GS-320HQ column. The flow rate was 1.0 mLÆmin )1 , and the absorbance at 260 nm of the effluent from the column was monitored. Polyacrylamide gel electrophoresis and molecular mass determination Native PAGE was carried out with 7.5% polyacrylamide gel according to the method of Davis [20]. Activity staining was carried out at 50 °C, as previously described [4]. SDS ⁄ PAGE was carried out using 15% polyacrylamide gel containing 0.1% SDS according to the method of Leammli [21]. The subunit molecular mass was determined using eight marker proteins (New England Biolabs). The molecular masses of the native enzymes were deter- mined by gel filtration column chromatography using Supe- rose 6 HR (Amersham Biosciences) with thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa) and ribonuclease A (13.7 kDa) serving as molecular standards (Amersham Bio- science). Analysis of the N-terminal amino-acid sequences The N-terminal amino-acid sequences of the enzymes were analyzed using an automated Edman degradation protein sequencer. After SDS ⁄ PAGE, the proteins were blotted onto polyvinylidene difluoride membranes and sequenced using a PPSQ-10 Protein Sequencer (Shimadzu, Kyoto, Japan). Acknowledgements This study was supported in part by the Pioneering Research Project in Biotechnology of the Ministry of Agriculture, Forestry and Fisheries of Japan and by the National Project on Protein Structural and Functional Analyses promoted by the Ministry of Education, Sci- ence, Sports, Culture, and Technology of Japan. 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