Download Purification and Some: Characteristics of a Monomeric Racemase

JOURNAL
OF BIOSCIENCE
AND BIOENGINEERING
Vol. 90, No. 3, 344-346. 2000
Purification and Some:Characteristics of a Monomeric Alanine
Racemase from an Extreme Thermophile,
Thermus thermophilus
TECK KEONG SEOW, 5 KENJI INAGAKI,* TAKESHI NAKAMURA, RITSUKO MAEDA,
TAKASH 1 TAMURA, AND HIDEHIKO TANAKA
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1-1-I Tsushima-Naka,
Okayama-shi, Okayama 700-8530, Japan
Received 11 April 2OOO/Accepted 16 June 2000
We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme
thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about
38,000. The enzyme was most active at pH 8 and 75X, and remained active after incubation at 80% for 30 min.
[Key words: alanine racemase, extreme thermophile,
Thermus thermophilus]
Since the first report of the enzyme in Streptococcus
faecalis (l), alanine racemase (EC 5.1.1 .l), which catalyzes the interconversion of the common L-stereoisomer
of alanine into D-alanine, has been investigated from
several different bacteria, including Bacillus stearothermophilus, a moderate thermophile (2), and Pseudomonas jluorescens, a psychrotroph (3). However, there
have been no reports of alanine racemase from extremely thermophilic and hyperthermophilic
bacteria that
thrive at temperatures beyond 65°C. Thermus thermophilus HB8 is an extreme thermophile that h.as long been
the subject of study of many biochemists, resulting in
the isolation of many useful enzymes (4-5). As there
had been no investigations on alanine racemase from
this bacterium, this project was initiated to screen for
and isolate the enzyme from T. thermophilus HB8.
Here, we report the purification and characterization of
alanine racemase from T. thermophilus HB8, which, to
our knowledge, is the first report of the enzlrme from an
extreme thermophile.
T. thermophilus HB8 was cultured aercsbically in a
medium (pH 7.5) containing 0.8% peptone, 0.4% yeast
extract, 0.2% NaCl, 0.35 mM CaC12, and 0.4 mM MgC&
at 75°C and harvested at 10-12 h after inoculation. Alanine racemase activity was routinely assayed in the D- to
L-alanine direction by a coupling reaction with L-alanine
dehydrogenase (2). The assay mixture (1 ml) contained
0.1 M 2-(cyclohexylamino)ethanesulphonic
acid (CHES)NaOH buffer (pH 9.0), 2.5 mM NAD+, 30mM D-alanine, 30 units of L-alanine dehydrogenase (‘EC 1.4.1 .l)
and an appropriate amount of enzyme solut:.on, and the
increase in absorbance at 340nm was monitored. Endpoint assay in the D- to L-alanine direction was also carried out for temperature and pH optima studies. The en-
zyme solution was incubated in 0.1 M CHES-NaOH
buffer (pH 9.0) and 30mM D-alanine for 30min in a
final volume of 1 ml. The reaction was terminated by
boiling the mixture for lOmin, and then the boiled mixture was incubated in 0.1 M CHES-NaOH
buffer
(pH 9.0), 2.5 mM NAD+, and 30 units of L-alanine dehydrogenase at 37°C for 1 h. The difference in the absorbance at 340nm was then compared with a standard
curve plotted from the results of the reaction between Lalanine and r.-alanine dehydrogenase to determine the
amount of L-alanine produced. One unit of alanine racemase activity was defined as the amount of enzyme catalyzing the formation of 1 /*mol of product per min at
25°C. Protein concentrations were determined by the
Bradford method (7) with bovine serum albumin used as
a standard. Protein elution patterns of column fractions
were measured by absorption at 280 nm.
The standard buffer, 10 mM 3-cyclohexylaminopropanesulphonic acid (CAPS)-NaOH buffer (pH 10.3) containing 0.01% 2-mercaptoethanol and 10 PM pyridoxal
5’-phosphate (PLP), was used throughout the purification procedures unless otherwise stated. All purification
procedures were carried out at 4°C. The harvested cells
(about 12Og) were suspended in 300ml of the standard buffer. After sonication, the suspension was centrifuged and the supernatant solution was dialyzed against
the standard buffer. The resultant cell-free extract was
brought to 60% saturation with ammonium sulphate
and the precipitate was dissolved in and dialyzed against
the same buffer. The dialyzed solution was loaded onto
a 275 ml ($5.0 x 14 cm) DEAE-Toyopearl 650M column
(Tosoh Corp., Tokyo) equilibrated with the standard
buffer. The column was washed with the standard buffer
and eluted with the same buffer containing 0.1 M NaCl
at a mean flow rate of 11 ml/min. The active fractions
were pooled, concentrated and dialyzed against the standard buffer containing 2.5 M NaCl. After dialysis, the
enzyme solution was applied onto a 275 ml ($5.0 x 14 cm)
Butyl-Toyopearl 650M column (Tosoh) equilibrated with
the dialysis buffer. After washing the column with the
dialysis buffer, the enzyme was eluted at a mean flow
rate of 11 ml/min with the standard buffer in a stepwise
NaCl gradient from 2.OM to 0.5 M. The active fractions were pooled, concentrated and dialyzed against the
* Corresponding author.
% Present address: Bioprocessing Technology Centre, National
University of Singapore, 10 Medical Drive, Singapore 117597, Singapore.
Abbreviations: CAPS, 3-cyclohexylaminopropanesulphonic
acid;
PLP, pyridoxal 5’-phosphate; CHES, 2-(cyclohexylamino)ethanesulphonic acid; FPLC, fast protein liquid chromatography; SDSPAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis;
MALDI-TOF,
matrix-assisted laser desorption/ioniz3tion
time of
flight; MS, mass spectrometer.
344
VOL.
90, 2000
NOTES
TABLE
1. Purification
of alanine racemase from T. thermophilus HB8
Total
activity
(unitsja
Cell-free extract
040% (NH4)$04
DEAE-Toyopearl650M
Butyl-Toyopearl650M
Superdex 200
Total
protein
(ma)
7900
5900
200
22
0.026
64
60
55
21
5.7
a The enzymatic activity was assayed in the
D-
Specific
activity
(units/mg)
Yield
(%)
0.0084
0.011
0.28
0.95
220
loo
94
84
33
8.9
Purification
index
Ifold)
1.0
1.3
34
120
28000
Ovalbumm
(43.W
Alanine
Rncemnse
P8,OM3
to L-alanine direction.
standard buffer. The resultant solution was subjected
to gel filtration using a Superdex 200 16/60 column
(Amersham Pharmacia Biotech AB, Uppsala, Sweden)
equilibrated with the standard buffer containing 0.2 M
NaCl connected to a fast protein liquid chromatography
(FPLC) system. El&on was carried out with the standard buffer containing 0.2 M NaCl at a flow rate of 0.5
ml/min. After the active fractions were pooled, concentrated and dialyzed against the standard buffer, the solution was frozen and stored at -20°C until further use.
The Superdex 200 16/60 column (Pharmacia) was calibrated using molecular weight markers (thyroglobulin, M,
669,000; ferritin, M, 440,000; catalase, M, 232,000; bovine serum albumin, M, 67,000; ovalbumin, M, 43,000;
chymotrypsinogen A, M, 25,000) to determine the molecular weight of the native enzyme. The relative mobility of the denatured enzyme after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
was compared with the relative mobility of protein standards (phosphorylase b, M, 94,000; bovine serum albumin, M, 67,000; ovalbumin, M, 43,000; carbonic anhydrase, M, 30,000; soybean trypsin inhibitor, Mr 20,100;
rw-lactalbumin, Mr 14,400) to estimate the subunit molecular weight of the enzyme. Matrix-assisted laser desorption/ionization
time of flight (MALDI-TOF)
mass
spectra were obtained on a Vision 2000 reflectron TOF
mass spectrometer (MS) (Finnigan MAT, Hemel Hempstead, England) using 2,5-dihydroxybenzoic acid as the
matrix. The external calibration of the MALDI mass
spectrum was established using ribonuclease A, chymotrypsinogen, and bovine serum albumin.
After fractionation using ammonium sulphate (O-60%
saturation), ion exchange using DEAE-Toyopearl 650M,
hydrophobic interaction chromatography by Butyl-Toyopearl 650M, and gel filtration using Superdex 200, we
purified the alanine racemase from T. thermophilus HB8
to homogeneity. As summarized in Table 1, the enzyme
was purified 28,000-fold with a yield of 8.9%, and
yielded a single band when resolved by SDS-PAGE.
From gel filtration, the alanine racemase was estimated
to have a molecular weight of 38,000, which is the same
as that obtained by SDS-PAGE. The similar values obtained suggest that the structure of the alanine racemase
from T. thermophilus HB8 is monomeric. Results of the
MALDI-TOF
MS analysis yielded a single-charged peak
with a molecular weight of 38,000, thereby confirming
the results obtained by SDS-PAGE (8). The molecular
weight of the subunit of the alanine racemase from T.
thermophilus HB8 is similar to that of other alanine racemases which are also about 40,000, such as Salmonella
typhimurium
345
(&& 39,000) (9), P. fluorescens (MC 38,000)
(3) and the fungus, Tolypocladium niveum (MI 37,000)
(10). The monomeric structure of the enzyme is similar
to that of the alanine racemase from S. typhimurium (9)
SDS-PAGE of alanine racemasefrom
T. thermophilus HB8
and possibly from S. faecalis (11, 12). Recent papers on
the alanine racemase from B. stearothermophilus (13-15)
indicated that the Tyr 265 residue from one subunit was
the second base of the two-base model proposed by
Adams (16), with the Lys 39 residue of the other subunit
being the first base. Both Tyr 265 and Lys 39 are conserved in all alanine racemases whose amino acid sequences are known (13). Given that there are very few
reports on monomeric alanine racemases and that all
recent reports described enzymes that are homodimeric
(2, 3, 10, 17), the discovery of a monomeric alanine racemase enzyme from T. thermophilus HB8 should aid in
the elucidation and a more thorough understanding of
the enzyme’s reaction mechanism.
As shown in Fig. 1, the alanine racemase from T. thermophilus HB8 exhibited a maximum activity at 75°C
and retained almost 100% of its activity even after being
incubated at 80°C for 30min, but lost most of its activity upon incubation for the same duration at 90°C. The
thermostability of the enzyme exceeded that from B.
stearothermophilus. The alanine racemase from T. thermophilus HB8 showed no loss of activity even after
being incubated for 30min at 8O”C, while the enzyme
from B. stearothermophilus only retained about 40% of
its original activity under similar conditions (2). The optimum temperature of 75°C for the alanine racemase
from T. thermophilus HB8 is the highest among all alanine racemases reported to date. This concurs with the
cultivation temperature of 75°C for the bacterium. As
a comparison, the enzyme from B. stearothermophilus
was reported to be most active at 60°C (2), while that
from Acidiphilium organovorum 13H, a mesophilic acidophile, demonstrated the highest activity at 50-60°C (17).
The alanine racemase from T. thermophilus HB8 was
also found to be most active at pH 8 and was stable
after 30 min of incubation in buffers from pH 5 and
above (data not shown).
The enzyme lost its activity upon dialysis against PLPfree standard buffer containing 10 mM hydroxylamine,
which suggests that the enzyme required PLP for its activity. Subsequent dialysis against 10 mM CAPS-NaOH
buffer (pH 10.3) containing 0.01% 2-mercaptoethanol
and 100,uM PLP restored the activity of the enzyme by
about 10%. The low recovery of enzymatic activity is believed to be due to the instability of the monomeric apo-
346
J. BIOSCI. BIOENG.,
SEOW ET AL.
REFERENCES
80
60
40
20
10 20 30 40 50 60 70 80 90 100
Temperature (T)
FIG. 1. Effect of temperature on alanine racemase from T. thermophilus HB8. The activity of the enzyme for the optimum temperature plot was assayed using the r.-alanine dehydrogenase end-point
assay method and is indicated by closed squares ( n ), The activity of
the enzyme for the thermostability plot was assayed using the L-alanine dehydrogenase coupling method after the enzyme samples were
incubated at their respective temperatures for 30 min, and the result is
represented by closed circles (0).
enzyme upon the removal of PLP. Efforts to detect the
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of other alanine racemases (2, 3, 9, 17) were unsuccessful. This was probably due to the very little amount of
purified enzyme obtained, which was only 0.026mg
(Table 1). Nevertheless, PLP requirement must be confirmed by further studies.
As demonstrated in this study, the enz:lme from T.
thermophilus HBS catalyzed the racemization of D- and
L-alanine. We were not able to investigate the catalytic
activity of the enzyme on other amino acids due to the
insufficient amount of the available purified enzyme.
Recognizing the significance of this information, further
study on the substrate specificity of the erzyme is currently underway.
The results of this study have shown that the alanine
racemase from T. thermophilus HBS is a very interesting
enzyme. We are presently cloning the alanine racemase
gene into Escherichia coli so that the enzyme may be
overexpressed for further characterization. This will contribute to a greater comprehension of the structure and
catalytic mechanism of alanine racemases in general.
We would like to thank Professor Tairo Oshima and Associate
Professor Akihiko Yamagishi of the Tokyo University of Pharmacy and Life Science for providing the T. thetmophilus HB8
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