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JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 98, No. 5, 391–393. 2004

Crystallographic Studies of Mycobacterium tuberculosisPolyphosphate/ATP-NAD Kinase

Complexed with NAD

SHIGETAROU MORI,1 MASAYUKI YAMASAKI,2 YUKIE MARUYAMA,1

KEIKO MOMMA,1 SHIGEYUKI KAWAI,1 WATARU HASHIMOTO,1

BUNZO MIKAMI,2 AND KOUSAKU MURATA1*

Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan1and

Laboratory of Food Quality Design and Development, Division of Agronomyand Horticultural Science, Graduate School of Agriculture,

Kyoto University, Uji, Kyoto 611-0011, Japan2

Received 13 August 2004/Accepted 27 August 2004

NAD kinase from Mycobacterium tuberculosis (Ppnk) uses ATP or inorganic polyphosphate[poly(P)]. Ppnk overexpressed in Escherichia coli was purified and crystallized in the presence ofNAD. Preliminary X-ray analysis of the resultant crystal indicate that the crystal belongs to hexa-gonal space group P6

222 and is holo-Ppnk complexed with NAD.

[Key words: NAD kinase, Mycobacterium tuberculosis, polyphosphate, NAD, ATP, crystallization]

In living organisms, NADP is formed through NAD phos-phorylation, which is catalyzed by NAD kinase (EC 2.7.1.23)in the presence of a phosphoryl donor (1). NAD kinase is re-garded as a key enzyme in NADP synthesis and, hence, innumerous cellular processes such as anabolic/biosyntheticpathways and protection against oxidative stress (1, 2). Weclarified the primary structures and properties of NAD ki-nases from bacteria, Micrococcus flavus (Mfnk) (3, 4), Myco-bacterium tuberculosis (Ppnk) (4), Escherichia coli (YfjB)(5), and yeast, Saccharomyces cerevisiae (Utr1p) (6). Fol-lowing our studies, other NAD kinases have reported forhuman (7), plant, Arabidopsis (NADK1 and NADK2) (8),yeast, S. cerevisiae (Pos5p and Yef1p) (2, 9, unpublisheddata), and bacteria, Bacillus subtilis (NadF) (10), and Sphin-gomonas sp. strain A1 (11). Of these, Mfnk, Ppnk, and NadFhave been shown to be inorganic polyphosphate [poly(P)]/ATP-NAD kinases (3, 4, 10), while YfjB, Utr1p, Yef1p,NadK, NADK1, and NADK2 are ATP-specific NAD ki-nases (ATP-NAD kinases) (5, 6, 8, 11, unpublished data).Inorganic polyphosphate [poly(P)]/ATP-NAD kinases useboth poly(P) and ATP, while ATP-NAD kinases use ATP butnot poly(P).

Poly(P) is a polymer of inorganic orthophosphate resi-dues linked by high-energy phosphoanhydride bonds, ap-proximately equivalent to that of ATP (12). Poly(P) is as-sumed to function as an ATP substitute in cells and may bean ancient energy carrier preceding ATP (12). Cost-wise,poly(P) is cheaper than ATP (13). Although we succeededin mass-producing NADP using poly(P) and poly(P)/ATP-

NAD kinase (Ppnk) from M. tuberculosis (13), the lack ofdifferent poly(P)-utilizing kinases prevented us from apply-ing poly(P) to the production of different biochemicals re-quiring ATP for production. Analyzing the crystal structureof Ppnk could clarify the poly(P) use of Ppnk and help de-termine biotechnological methods to confer on differentATP-specific kinases the ability to use poly(P). The struc-tural analysis of Ppnk would thus be indispensable in apply-ing poly(P) to producing a variety of biochemicals.

We reported preliminary crystallographic data on Ppnk(14). Although the crystal structure of apo-Ppnk has beenroughly determined (15), its coordinates have not yet beenavailable. Substrate [NAD, ATP, and poly(P)]-binding sitesand catalytic sites of Ppnk also have yet to be clarified,since no crystal structural information on holo-Ppnk com-plexed with a substrate has been determined. The two sub-strates of Ppnk and other NAD kinases, a phosphoryl donor(ATP) and acceptor (NAD), contain ADP, in which the 2�hydroxyl group of ribose in the ADP moiety of NAD isphosphorylated by NAD kinase, resulting in NADP. Theclarification of substrate-binding sites of Ppnk is importantto understanding how Ppnk discriminates between two ADPmoieties in ATP and NAD, so we attempted to obtain theholocrystal of Ppnk complexed the substrate.

Ppnk was purified from the cell extract of recombinant E.coli as described elsewhere (4), but using KNDE (10 mMpotassium phosphate, pH 7.0, 0.2 mM NAD, 1 mM EDTA,and 0.5 mM dithiothreitol) as a basal buffer. Using EDTAinhibited proteolytic degradation of Ppnk and enabled us toobtain purified Ppnk. Ppnk was then concentrated by ultra-filtration with Centriprep (Millipore, Bedford, MA, USA) toyield a final concentration of 10 mg/ml. Protein concentra-tion was determined by the method of Bradford (16). Ppnk

* Corresponding author. e-mail: kmurata@kais.kyoto-u.ac.jpphone: +81-(0)774-38-3766 fax: +81-(0)774-38-3767The last two authors contributed equally to this work.

MORI ET AL. J. BIOSCI. BIOENG.,392

was crystallized by hanging-drop vapor diffusion on Linbrotissue-culture plates (ICN Biomedicals, Auora, OH, USA).The Ppnk crystal was prepared in a hanging drop (6 �l),mixed with 3 �l each of the reservoir solution (1.6 M ammo-nium sulfate, 0.1 M HEPES–Na, pH 8.0, and 5 mM NAD)and Ppnk (10 mg/ml) in KNDE on a siliconized coverslipover 1.0 ml of the same reservoir solution. The conditiondiffered from that in which the apo-Ppnk crystal was ob-tained (14).

A prismatic colorless Ppnk crystal formed in about twoweeks at 20�C, growing to a maximum 0.2 mm under thecondition containing NAD (Fig. 1). To mitigate damage andmeasure higher resolution data, we collected data with asynchrotron at low temperature (100 K). Since the crystalfroze in the absence of cryoprotectant, we soaked it incryoprotectant solution (2.0 M ammonium sulfate, 0.1 MHEPES–Na, pH 8.0, 5 mM NAD, and 25% glycerol) for10 min. Diffraction data up to 2.70 Å was collected usingsynchrotron radiation at a wavelength of 1.0000 Å at theBL-38B1 station of SPring-8 in Hyogo, Japan. The obtaineddata were processed, merged, and scaled using programpackage HKL 2000 (DENZO and SCALEPACK) (17), andtruncated with the Xtalview program (18).

Preliminary crystal characterization indicated it belongedto hexagonal space group P6222 with unit cell dimensionsof a�b�110.4, c�108.9 Å. Among all reflections (93,585)observed, 11,192 independent reflections were obtained with

an Rmerge of 6.5%. Data exhibited completeness of 98.8% atup to 2.70 Å resolution. Data collection statistics for the crys-tal are summarized in Table 1. Ppnk is a tetramer of identi-cal subunits with a molecular mass of about 35 kDa (4). Onesubunit of Ppnk per asymmetric unit yields a VM of 2.76Å/Da and a solvent content of 56%. VM and solvent contentlay within the range usually found for protein crystals (19).The crystallographic data we obtained on the Ppnk crystalwas distinct from that on apo-Ppnk reported previously (14).The apo-Ppnk crystal belonged to monoclinic space groupC2 with unit cell parameters of a�140.0, b�69.3, c�106.3 Å,and ��130.1� (14).

Since the crystal presented here was obtained under acondition containing NAD, we expected that the crystal washolo-Ppnk complexed with NAD. When we determined thestructure of the expected holo-Ppnk by molecular replace-ment method using apo-Ppnk structure as a model, wefound a electron density map, which corresponded well tothe molecular structure of NAD (Fig. 2). Hence, the crystalpresented here is considered to be holo-Ppnk crystal com-plexed with NAD.

In primary structures of NAD kinases, two conserved re-gions (I and II) were found (5, 20) (Fig. 3). Our preliminaryanalysis of the holo-Ppnk crystal complexed with NAD in-dicated that both the two conserved regions and another re-gion, which we specify as region III in Fig. 3, participated in

FIG. 1. Ppnk crystal complexed with NAD.

TABLE 1. Data collected for the Ppnk crystal

Wave length (Å) 1.0

Resolution limit (Å) 50.0�2.7 (2.80�2.70)Space group P6222Unit cell dimensions (Å) a�b�110.4, c�108.9Subunits/Asymmetric unit 1Measured reflections 93585 (5243)Unique reflections 11192 (1070)Redundancy 8.4 (4.9)Completeness (%) 98.8 (98.1)Rmerge (%) 6.5 (35.0)

Values in parentheses refer to data in the highest resolution shell.

FIG. 2. Stereodiagram of the electron density map of NAD and the surrounding amino acid residues. The 2|Fo|� |Fc| map using model omit-ting NAD is shown. NAD is shown as a red stick model. Amino acid residues are indicated as cyan stick models. This figure was prepared usingthe program TURBO-FRODO (AFMB-CNRS, Marseille, France).

NOTESVOL. 98, 2004 393

NAD binding. We are now in the process of determining thecrystal structure of holo-Ppnk complexed with NAD andpreparing the holo-Ppnk crystal complexed with ATP orpoly(P). Crystallographic studies using holocrystals of Ppnkare expected to enable poly(P) to be applied to the produc-tion of a variety of biochemicals.

This work was supported in part by a grant in aid from the Min-istry of Education, Culture, Sports, Science, and Technology of Ja-pan (no. 15780212), and by the Program for Promotion of BasicResearch Activities for Innovative Biosciences (PROBRAIN).

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FIG. 3. Multiple alignment of primary structures of poly(P)/ATP- and ATP-NAD kinases. The primary structures of NAD kinases, for whichphosphoryl donor [poly(P) and ATP] specificities were experimentally reported, are aligned using ClustalW (21). Poly(P)/ATP kinases are as fol-lows: Ppnk [307 residues], M. tuberculosis (AB044336-1) (4), Mfnk [362], M. flavus (AB070351) (3), and NadF [266], B. subtilis (SwissProt:O31612) (10). ATP-NAD kinases are as follows: YfjB [292], E. coli (D90888-18) (5), NadK [298], Sphingomonas sp. A1 (AB127931) (11), Utr1p[530] (Z49549-1) (6), Yef1p [495] (U18779-13) (unpublished data), Pos5p (Z73544-1) [414] (2, 9), S. cerevisiae, NADK1 [524] (AY383545-1),and NADK2 [985] (AF337912), Arabidopsis (8). Genbank (http://www.genome.ad.jp/dbget-bin/www_bfind/genbank-today) IDs are indicated ex-cept for NadF, which is indicated by its SwissProt (http://www.genome.ad.jp/dbget-bin/www_bfind/swissprot-today) ID. Numbers of residues foreach enzyme are specified. Identical residues are denoted by an asterisk (*), strongly conserved residues by a colon (:), and weakly conserved resi-dues by a period (.). Conserved regions I, II, and III are specified above alignment.