Biosci. Biotechnol. Biochem., 74 (9), 1936–1939, 2010
Note
Molecular Cloning and Characterization of ꢀ-Glutamyltranspeptidase
from Pseudomonas nitroreducens IFO12694
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1;
Masashi IMAOKA,1 Shigekazu YANO,1 Masashi OKUMURA,1 Takao HIBI,2 and Mamoru WAKAYAMA
1Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
2Department of Bioscience, Faculty of Biotechnology, Fukui Prefectural University,
Eiheiji-cho, Yoshida-gun, Fukui 910-1195, Japan
Received March 18, 2010; Accepted June 1, 2010; Online Publication, September 7, 2010
ꢀ-Glutamyltranspeptidase from Pseudomonas nitro-
reducens IFO12694 (PnGGT) exhibited higher hydro-
lytic activity than transfer activity, as compared with
other ꢀ-glutamyltranspeptidases (GGTs). PnGGT
showed little activity towards most of L-amino acids
and towards glycyl-glycine, which is often used as a
standard ꢀ-glutamyl accepter in GGT transfer reactions.
The preferred substrates for PnGGT as a ꢀ-glutamyl
accepter were amines such as methylamine, ethylamine,
and isopropylamine.
sion in E. coli and the properties of the enzyme,
especially its novel substrate specificity, are reported.
E. coli JM109 was used as a host strain in preparing
the plasmid. E. coli Rosetta-gami B (DE3) and pET22-b
from Novagen (Madison, WI) were used for gene
expression. Judging from the N-terminal amino acid
sequences of large and small fragments of the purified
enzyme and the nucleotide sequences of the other
microbial GGTs, it is thought that its gene codes a signal
peptide, a large subunit and a small subunit in a single
peptide. At first, then, part of the PnGGT gene was
amplified from P. nitroreducens genomic DNA using
forward primer 50-gtcaccctcgacggcggcgcggtt-30 and
reverse primer 50-gtcgacgatggagaagtgcgtggt-30, designed
based on N-terminal amino acid sequences (large frag-
ment, VTLDGGAVAAPDQYGAKVAA; small frag-
ment, TTHFSIVDKDGNAVSNTYTL) and the nucleo-
tide sequences of highly conserved regions among
Pseudomonas GGTs. To obtain DNA fragments corre-
sponding to the upstream and the downstream regions of
the partial PnGGT gene, an LA-PCR in vitro cloning kit
(Takara, Shiga, Japan) was used (BamHI and EcoRI
cassettes). The determined upstream and downstream
sequences of the partial PnGGT gene allowed us to
design PCR primers, forward primer 50-taaggaagtcatatg-
cgcgtgttccacttc-30 (underlined region, NdeI restriction
site) and reverse primer 50-acggtgcgaattccggggcttaggg-
tttga-30 (underlined region, EcoRI restriction site), for
amplification of the complete PnGGT gene. PCR was
performed in a reaction mixture containing 10 ng of
genome template DNA, 50 pmol of each primer, 10 nmol
of each dNTP, and 2.5 U of ExTaq DNA polymerase
(50 ml in ExTaq buffer). Thermal cycling was 1 cycle of
94 ꢀC for 1 min, followed by 30 cycles of 98 ꢀC for 10 s,
55 ꢀC for 30 s, and 72 ꢀC for 90 s. A PCR product was
cloned into pET22-b and its nucleotide sequence was
determined. E. coli Rosetta-gami B (DE3) harboring
pPnGGT2 was cultured for PnGGT production.
Key words: Pseudomonas nitroreducens; ꢀ-glutamyl-
transpeptidase; ꢀ-glutamylmethylamide
ꢀ-Glutamyltranspeptidase (GGT) catalyzes the trans-
fer of the ꢀ-glutamyl moiety from ꢀ-glutamyl com-
pounds, such as glutathione, to amino acids and
peptides, as well as the hydrolysis of ꢀ-glutamyl
compounds. It is thought to play key roles in glutathione
metabolism in both prokaryotic and eukaryotic organ-
isms. Since its discovery in the sheep kidney,1) it has
been isolated and characterized from various sources
such as humans,2) rats,3) radishes,4) fungi,5) Escherichia
coli,6) Helicobacter pylori,7) and Bacillus subtilis.8)
Enzymatic synthesis of useful ꢀ-glutamyl compounds
such as ꢀ-L-glutamyltaurine and ꢀ-glutamylethylamide
(theanine) using GGT has been reported.9,10) Efficient
theanine synthesis from glutamine and ethylamine by
immobilized cells of P. nitroreducens has also been
reported.11) The enzyme catalyzing this transfer reaction
is reported to be glutaminase based on a classification by
Hartman.12,13) To ensure high production and for
clarification of the reaction mechanism of this enzyme,
molecular cloning of its gene is a prerequisite. Analysis
of the N-terminal regions of the purified enzyme
(heterodimeric polypeptides) clarified that the N-termi-
nal amino acid sequences of the enzyme have high
homology to those of microbial GGTs, whose hetero-
dimeric polypeptides are generated by autocatalytic
processing as found in the N-terminal nucleophile
hydrolase superfamily. Consequently, the enzyme clas-
sified as a glutaminase based on Hartman’s classification
structurally belongs to GGTs. The DNA region encoding
it was amplified by PCR using primers designed based
on these N-terminal amino acid sequences and the
nucleotide sequences of the highly conserved regions
among microbial GGTs. In this study, PnGGT expres-
GGT activity was measured as follows: For hydrolytic
activity, the reaction mixture contained 2.5 mM of ꢀ-L-
glutamyl-p-nitroanilide (ꢀ-GlupNA), 100 mM imidazole
buffer (pH 9.0), and the enzyme in a final volume of
1 ml. The reaction was terminated by adding 0.5 ml of
10% acetic acid after incubation at 30 ꢀC for 10–20 min.
p-Nitroaniline formation was monitored at 410 nm. One
unit of enzyme in the hydrolytic reaction was defined as
the amount required to catalyze the formation of 1 mmol
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