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

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

*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

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].

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__________________________________________

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]