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ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(7): 666–670                                    CN 31-1300/Q

 

Short Communication

Expression, Purification, and Characterization of Recombinant Saccharomyces cerevisiae Adenosine Kinase

LV Xiao-Bing, WU Hai-Zhen, YE Jiang, FAN Yi, ZHANG Hui-Zhan*

( State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China )

 

Abstract       Adenosine kinase (AK), a key enzyme in the regulation of the cellular concentrations of adenosine (A), is an important physiological effector of many cells and tissues. In this article, we reported that ak, which encoded adenosine kinase, was cloned from Saccharomyces cerevisiae, sequenced, and overexpressed in E.coli using the pET16b expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of S.cerevisiae ak revealed Km values of (3.5±0.2) μmol/L for adenosine and (100.0±11.0) μmol/L for ATP, with kcat of (1530±20) min－1 for adenosine and (1448±25) min－1 for ATP. The determination of the Km value for other nucleosides and deoxynucleoside indicated that the nucleoside specificity of this enzyme from yeast was quite high.

 

Key words     adenosine kinase; protein purification; enzyme kinetics; Saccharomyces cerevisiae

 

Adenosine kinase (AK) (ATP: adenosine 5′ phosphotransferase, EC 2.7.1.20) with broad tissue and species distribution has been isolated from certain microorganisms[1], yeast[2], Toxoplasma gondii[3], and various mammalian sources[4－8]. AK is a key enzyme in the regulation of the intracellular level of adenosine[9－11]. It catalyzes the phosphorylation of adenosine to AMP, preferentially using ATP as the phosphate source. Magnesium is also required for this reaction and is probably associated with the substrate nucleotide[4,9]. The pH optimum of the enzyme was a function of the ratio of ATP to magnesium concentration[12]. AK is responsible for the activation of many pharmacologically active nucleosides, including tubercidin, formycin, ribavivin, pyrazofurin and 6-(methylmercapto) purine riboside[6]. The loss of AK activity has been implicated as a mechanism of cellular resistance to the pharmacologic effects of these nucleoside analogs[13].

Previous analysis of yeast extracts and purified AK protein from dried brewer’s yeast[2] have demonstrated that the nucleoside specificity of the enzyme from yeast is rather restricted. More detailed biochemical studies have not been reported probably because of the low production of purified native yeast AK, but this difficulty has recently been circumvented by the molecular cloning of the yeast ak. We report here on the overexpression of S.cerevisiae ak in E.coli, purification of the resulting protein to homogeneity, and biochemical characterization of this recombinant enzyme.

 

1    Materials and Methods

1.1   Materials

1.1.1       Reagents        DNA restriction enzymes and T4 DNA ligase were purchased from TaKaRa Biotechnology (Dalian, China) Co., Ltd.. Isopropylthio-β-D-galactoside (IPTG) was purchased from Promega. Five kinds of nucleosides and 2′-deoxyadenosine were purchased from Biosia, Sigma, and Fluka. DEAE-Sepharose was purchased from the Amersham Biosciences. All other chemicals were of analytical grade.

1.1.2       E.coli strains and culture conditions     E.coli strains JM83 and BL21(DE3) were used for the general construction of plasmids[14]. Plasmids pUCm-T and pET16b were used as vectors.

 

Table 1 Strains and plasmids

Strain and plasmid

Characteristic

Source

E.coli

JM83

F－, ara, Δ(lac-proAB), rpsL, (strR),
φ80ΔlacZΔM15

This laboratory

BL21(DE3)

F－, ompT, hsdSB, (r－B,m－B), dem,
gal, λ(DE3)

This laboratory

Yeast

 

S.cerevisiae

This laboratory

Plasmids

pUCm-T

ampR

TaKaRa

pET16b

ampR, T7 promoter

This laboratory

 

The strains and plasmids used in the study are listed in Table 1. All E.coli strains were grown in Luria-bertani (LB) medium and LB agar (18 g/L) plates supplemented with ampicillin (100 mg/L) when needed[14].

 

1.2 Methods

1.2.1       Cloning and expression of the S.cerevisiae ak in E.coli       We found that an amino acid sequence in S.cerevisiae was highly homologous with several sequences of AK in various organisms (using the information from GenBank and Blast). Primers: 5′-GCAACCATGGCCGCACCATTGGTAGTATTGGG-3′, and 5′-AAGAATCTATTTAGAGTAAGATATTTTTTCGG-3′ were designed for PCR with S. cerevisiae chromosome as the template. The product was subcloned into pMD18-T vector, sequenced by the dideoxynucleotide chain-termination method, and transferred as an NcoI-BamHI fragment into bacterial expression vector pET16b. E.coli BL21(DE3) transformant was grown at 37 ℃ in LB media containing 100 mg/L ampicillin for 4 h and induction with 1 mmol/L IPTG was carried out for 4 h.

1.2.2       Purification of recombinant S.cerevisiae ak BL21(DE3) harboring ak-pET16b were grown to late lag phase, then the cells were harvested by centrifugation at 8 000 r/min for 10 min and suspended in TMD100 buffer[15][100 mmol/L Tris·HCl(pH 7.5), 5 mmol /L MgCl2, 2 mmol/L dithiothreitol (DTT)]. The cells were ruptured by sonication with an ultrasonic disintegrator. To concentrate the protein, solid ammonium sulfate (70%－90%) was added to the crude extract allowing 30 min for precipitation at 0 ℃, then centrifuged at 15 000 r/min for 30 min. The protein pellets were suspended in TD100[15][100 mmol/L Tris·HCl(pH 7.5), 2 mmol/L DTT]. Subsequently the enzymatic solution was dialyzed against TD100 buffer until complete equilibration was achieved. The resulting solution was further purified by a modification of published procedures[15] using DEAE-Sepharose FF chromatography. The purified AK protein was stored at －20 ℃ with 10% glycerol.

1.2.3       Protein concentration assay   Protein concentration was determined by absorbance at 280 nm and at 260 nm according to the equation [16]:

 

[Protein] (g/L)= 1.45×A280－0.74×A260.

 

1.2.4       AK assay        AK activity was measured by HPLC[17－20] (HPLC, Agilent 1100, Agilent Technologied CO., American) assays. A standard reaction mixture (100 μl) contained 50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L DTT, 2.5 mmol/L ATP and 2.5 mmol/L adenosine. The reaction was initiated by the addition of 0.1－2 μg of protein and terminated by heating in boiling water for 3 min. The reaction was conducted at 37 ℃. Samples were taken at 2 min intervals over the course of a 30 min assay, and then 20 μl of reaction mixture was infected into HPLC to detect the content of adenosine. According to the decrease of adenosine, one unit of adenosine kinase catalyzes the phosphorylation of 1 μmol adenosine per min under these conditions.

The apparent Km value for adenosine was determined at 1 mmol/L ATP using adenosine concentrations ranging from 1 μmol/L to 20 μmol/L. The apparent Km value for ATP was obtained at 20 μmol/L adenosine with ATP concentrations ranging from 50 μmol/L to 800 μmol/L. Kinetic parameters for other substrates of AK were determined at 1 mmol/L ATP with the following concentration ranges for other substrate: 0.5－3 mmol/L 2′-deoxyadenosine (2′-dA); 0.5－3 mmol/L inosine (I); 2－5 mmol/L guanosine (G); 0.5－3 mmol/L uridine (U); 0.5－3 mmol/L cytidine (C). Km and kcat values were calculated after Hanes-Woolf analysis of initial rate data.

 

2    Results

2.1   Expression of ak in E.coli BL21(DE3)

Comparison of the deduced AK amino acid sequence with the Swiss-Prot protein sequence database revealed 54% identity with rat brain adenosine kinase, 45% identity with human adenosine kinase, and 47% identity with T. gondii adenosine kinase. These comparisons suggested that ak encoded an adenosine kinase.

To confirm that ak encodes an adenosine kinase, we cloned ak into a pET16b vector to make it express in E.coli BL21(DE3).

E.coli harboring the ak-pET16b vector produced AK protein at a high level (25% in total amount of proteins) (Fig.1). Conventional purification methods were adopted to successfully purify S.cerevisiae ak to apparent homogeneity, as shown by SDS-PAGE (Fig.2). The anion exchange chromatography resulted in an overall twenty-fold purification of the enzyme.

 


Fig.1 The SDS-PAGE analysis of expression products of ak in E.coli BL21 (DE3)

1, E.coli BL21 (DE3) without induction; 2, E.coli BL21 (DE3) induced for 1 h; 3, E.coli BL21 (DE3) harboring pET16b without induction; 4, E.coli BL21 (DE3) harboring pET16b induced for 1 h; 5, standard protein molecular weight; 6, E.coli BL21 (DE3) harboring ak-pET16b without induction; 7, E.coli BL21 (DE3) harboring ak-pET16b induced for 1 h; 8, E.coli BL21 (DE3) harboring ak-pET16b induced for 2 h; 9, E.coli BL21 (DE3) harboring ak-pET16b induced for 3 h; 10, E.coli BL21 (DE3) harboring ak-pET16b induced for 4 h.

 


Fig.2 Recombinant enzyme was analyzed by SDS-PAGE on 15% slab gels

1, molecular weight marker; 2, soluble fraction of crude bacterial lysate; 3, ammonium sulfate precipitation fraction; 4, DEAE column eluation at 60－80 mmol/L NaCl.

 

Recombinant S.cerevisiae AK migrates on SDS-PAGE as a single band at ≈37 kD (Fig.2, lane 4), which is in close agreement with the 36.4 kD size predicted from the amino acid sequence.

The results of the steps of purification are compiled in Table 2.

 

Table 2 Summary of purification procedure

Step

Total volume
(ml)

Activity
(u/ml)

Total
activity (u)

Protein*
(g/L)

Specif
activity
(u/mg)

Recovery
(%)


Purification
(fold)

Crude extract

200

16.4

3284

3.22

5.1

100

1

Ammonium sulfate (70%－90%)

60

25.9

1555

0.73

35.5

47.3

7

DEAE Sepharose (NaCl fraction)

20

56

1130

0.56

100

34.4

20

*Protein concentration was determined by the absorbance at 280 nm and 260 nm.

 

2.2 AK activity assay

AK activity was measured by assaying the consumption of adenosine using HPLC. Rates were linear over the course of these assays, and <10% of total substrate in the reaction mix was utilized (Fig. 3).

 


Fig.3 Adenosine kinase activity assay was performed as described in Materials and Methods

 

Analysis of steady-state kinetics for the native substrates of S.cerevisiae ak indicates a Km values of (3.5±0.2) μmol/L for adenosine and (100.0±11.0) μmol/L for ATP (Fig. 4). The turnover number of the enzyme was calculated to be (1530±20) min－1 and (1448±25) min－1 at saturating adenosine and ATP concentrations respectively.

 


Fig.4 Kinetic analysis of recombinant S.cerevisiae AK activity

Kinetic analysis of S.cerevisiae AK activity was determined at various substrate concentrations as described in Materials and Method, and Hanes-Woolf analysis was used to determining kinetic parameters. (A) adenosine kinetics measured at 1 mmol/L ATP. (B) ATP kinetics measured at 20 μmol/L adenosine. Data presented is the x±s from at least three experiments.

 

The ability of substrate analogs to interfere with adenosine phosphorylation by purified recombinant S.cerevisiae AK suggests that these compounds can themselves serve as enzyme substrates. AK recognition of other four nucleosides and one deoxynucleoside as substrates were therefore assayed by HPLC. As shown in Table 3, all four analogs were phosphorylated by S.cerevisiae AK. The Km values for these substrates were much greater than the Km values for adenosine and ATP, however, and the kcat values were significantly reduced about 25, 44, 51, 15, 25 times for 2′-dA , I, G, U and C, respectively (Table 3).

 

Table 3 Summary of kinetic parameters for substrates of the S.cerevisiae AK

Substrate

A

2′-dA

I

G

U

C

ATP

Km(μmol/L)

3.5±0.2

660±35

800±32

2240±55

860±25

900±42

100.0±11.0

kcat(min－1)

1530±20

60±30

35±28

30±25

100±35

61±30

1530±20

kcat/Km

437.1

0.09

0.04

0.01

0.12

0.07

15.3

 

3 Discussion

E. coli was chosen for the expression of S.cerevisiae AK mainly because it does not contain any endogenous AK activity[21]. This fact was verified in our experiment, thus the observed AK activity could be ascribed solely to the heterologous expression of the enzyme.

It was reported that the kinetic properties of adenosine kinase from a variety of sources had been studied using radiometric assays[6,22]. In this study, we adopted HPLC assays, which is a new application in measuring adenosine kinase activity. The results indicate that the method is simple, rapid, sensitive and reproducible.

Kinetic studies on AK reveal the Km value for S.cerevisiae adenosine is significantly higher than values measured for recombinant or native human AK (41 nmol/L and 57 nmol/L, respectively)[23] and close to value measured for recombinant Toxoplasma gondii AK [(1.9±0.6) μmol/L and (54.3±19.3) μmol/L, respectively][15], but Michaelis constants for other mammalian AK enzyme range from 40 nmol/L to 20 μmol/L[24]. The S.cerevisiae enzyme shows a strict specificity for adenosine among naturally occurring nucleosides. Deoxyribonucleosides are only weakly accepted as substrates. This result is identical with the report about the adenosine kinase in mammalian[6].

The purified protein was analyzed in an in vitro enzymatic assay as described in the previous section. Initial attempts to assay of recombinant S.cerevisiae AK were unsuccessful, as the dithiothreitol (DTT) was not added during the purification and reaction procedure. The activity can be fully recovered in the presence of excess reducing agent. This indicates the presence of a thiol group at or near the active site of the enzymes.

Large quantities of purified enzyme is also a prerequisite for determination of the tertiary structure of S.cerevisiae ak, a task which should be expedited by the recent determination of the X-ray crystal structure of recombinant human AK[25,26]. The purified AK from microbial and mammalian origin share several characteristic properties such as a relatively low molecular weigh and a rather pronounced instability[27].

 

References

1     Datta AK, Bhaumik D, Chatterjee R. Isolation and characterization of adenosine kinase from Leishmania donovani. J Biol Chem, 1987, 262: 5515－5521

2     Leibach TK, Spiess GL, Neudecker TJ, Peschke GJ, Puchwein G, Hartmann GR. Purification and properties of adenosine kinase from dried brewer’s yeast. Hoppe Seylers Z Physio Chem, 1971, 352: 328－344

3     Recacha R, Talalaev A, DeLucas LJ, Chattopadhyay D. Toxoplasma gondii adenosine kinase: Expression, purification, characterization, crystallization and preliminary crystallographic analysis. Acta Crystallogr D Biol Crystallogr, 2000, 56: 76－78

4     Pallela TD, Andres CM, Fox IH. Human placental adenosine kinase. Kinetic mechanism and inhibition. J Biol Chem, 1980, 255: 5264－5269

5     Yamada, Goto H, Ogasawara N. Differences of adenosine kinases from various mammalian tissues. Comp Biochem Physio B, 1982, 71(3): 367－372

6     Miller RL, Adamczyk DL, Miller WH. Adenosine kinase from rabbit liver. I. Purification by affinity chromatography and properties. J Biol Chem, 1979, 254: 2339－2345

7     Rotllan P, Miras Portugal MT. Adenosine kinase from bovine adrenal medulla. Eur J Biochem, 1985, 151: 365－371

8     Sakowicz M, Grden M, Pawelczyk T. Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity. Acta Biochim Pol, 2001, 48(3): 745－754

9     Fox IH, Kelley WN. The role of adenosine and 2′-deoxyadenosine in mammalian cells. Annu Rev Biochem, 1978, 47: 655－686

10    Decking UK, Schlieper G, Kroll K, Schrader J. Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release. Cirs Res, 1997, 81: 154－164

11    Lloyd HG, Fredholm BB. Involvement of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration in rat hippocampal slices. Neurochem Int, 1995, 26(4): 387－395

12   Maj M, Singh B, Gupta RS. The influence of inorganic phosphate on the activity of adenosine kinase. Biochim Biophys Acta, 2000, 1476: 33－42

13    Bennett LL Jr, Schnebli HP, Vail MH, Allan PW, Montgomery JA. Purine ribonucleoside kinase activity and resistance to some analogs of adenosine. Mol Pharmacol, 1966, 2: 432－443

14    Smabrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 1989

15    Darling JA, Sullivan WJ Jr, Carter D, Ullman B, Roos DS. Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase. Mol Biochem Parasitol, 1999, 103: 15－23

16    Zhang LX, Zhang TF, Li LY. Experimental Methods and Techniques in Biochemistry, 2nd ed. Beijing: Higher Education Press, 1997

17    Zhao J, Todd B, Fleet GH. Seperation of ribonucleotides, ribonucleosides, deoxyribonucleotides, deoxyribonucleosides and bases by reversed-phase high-performance liquid chromatography. J Chromatogr A, 1994, 673: 167－171

18    Huang XL, Li KD, Chen YH. Seperation of fifteen hydrolysates of nucleic acid by high performance liquid chromatography and application in determining yeast extract. Chinese J Anal Chem, 2000, 28(12): 1504－1507

19    Bohacek J. Isocratic seperation of DNA bases and deoxyribonucleosides by high-performance liquid chromatography on anex OSTION. Anal Biochem, 1979, 94: 237－241

20    Miller AA, Benvenuto JA, Loo TL. Comparison of the reversed-phase high-performance liquid chromatographic seperation of fluoropyrimides, pyrimidines, and purines. J Chromatogr, 1982, 228: 165－176

21    Neidhardt FC ed. Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Washington DC: Am Soc Microbiol, 1987, Vol(I): 445－473

22    Mimouni M, Bontemps F, van den Berghe G. Kinetic studies of rat liver adenosine kinase. Explanation of exchange reaction between adenosine and AMP. J Biol Chem, 1994, 269(27): 17820－17825

23    Spychala J, Datta NS, Wakabayashi K, Datta M, Fox IH, Gribbin T, Mitchell BS. Cloning of human adenosine kinase cDNA: Sequence similarity to microbial ribokinases and fructokinases. Proc Natl Acad Sci USA, 1996, 93: 1232－1237

24    Chang CH, Brockman RW, Bennett LL Jr. Adenosine kinase from L1210 cells. Purification and some properties of the enzyme. J Biol Chem, 1980, 255: 2366－2371

25    Mathews II, Erion MD, Ealick SE. Structure of human adenosine kinase at 1.5  resolution. Biochemistry, 1998, 37: 15607－15620

26    McNally T, Helfrich RJ, Cowart M, Dorwin SA, Meuth JL, Idler KB, Klute KA et al. Cloning and expression of the adenosine kinase gene from rat and human tissues. Biochem Biophys Res Commun, 1997, 231: 645－650

27    Luft BJ, Brooks RG. Conley FK, McCabe RE, Remington JS. Toxoplasmic encephalitis in patients with acquired immune deficiency syndrome. JAMA, 1984, 252: 913－917

__________________________________________

Received: March 24, 2003     Accepted: April 29, 2003

This work was supported by a grant from Star Lake Co. Inc., Zhao qing, Guang dong, China

*Corresponding author: Tel, 86-21-64252515; Fax, 86-21-64252255; e-mail, [email protected]