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A. SENOO et al.
as the dry gas, and the capillary was set at 3.5 kV. The
flow rate was maintained at 10 ml/min, and 5 mM
ammonium acetate dissolved in 50% methanol was
used as the sheath liquid to detect molecular-related ions
from m=z 70 to 500 in positive mode.
Five enzymes (SMU.1321c from Streptococcus
mutans, Plu1218 from Photorhabdus luminescence
subsp. laumondii, TDE2209 from Treponema denticola,
Aple02000835 from Actinobacillus pleuropneumoniae,
and SP0885 from Streptococcus pneumoniae) were
found to possess Lal activity (Table 2). It was found
that each enzyme catalyzed the formation of dipeptides
that could not be synthesized by known Lals. The
substrate specificities of the enzymes are summarized
in Fig. 1.
SMU.1321c: Dipeptides formation was confirmed in
27 combinations of amino acid by CE-MS analysis
(Fig. 1d). Aromatic amino acids (Phe, Tyr, and Trp) and
His were preferred as substrates. The formation of
dipeptides by combination of (Gly, Tyr), (Leu, Trp),
(Met, Trp), and (Trp, Tyr) by Lals was not confirmed.
Plu1218: Dipeptide formation was confirmed in two
combinations of substrates, (Gln, Phe) and (Gln, Glu)
(Fig. 1e). Dipeptide formation from Gln and Glu by Lals
was not confirmed.
assumed that HMM-based profile analysis is an alter-
native tool in the search for a novel enzyme.
We obtained only nine soluble recombinant proteins
out of 18 selected candidate genes, and thus Lal
homologs easily form inclusion bodies under strong
expression. There is room for improvement in expres-
sion, although we adopted two methods of protein
expression, the pET and the pCold system.
Our results indicate that Lals are widely distributed
in bacteria such as Streptocccus, Photorhabdus, Trepo-
nema, and Actinobacillus, although their functions
remain unclear. It has been reported that YwfE is the
enzyme involved in the biosynthesis of bacilysin, a
dipeptidic antibiotic produced by B. subtilis.13) Bacilysin
contains L-Ala at the N-terminus and a non-proteinogenic
amino acid, L-anticapsin, at the C-terminus, and YwfE
catalyzes the ligation of L-Ala and L-anticapsin. Recently
another Lal, RizA in B. subtilis NBRC3134, related to
the synthesis of peptide-antibiotic Rhizoctin, was iden-
tified.7) According to these results, Lals might be
involved in the biosynthesis of various unknown
peptidyl compounds in bacteria.
Acknowledgments
TDE2209: Dipeptide formation was confirmed in
three combinations of substrates, (Gly, Pro), (His, Pro),
and (Cys, Glu) (Fig. 1f). Dipeptide formation from Gly
and Pro by Lals was not confirmed.
We thank Ritsuko Katahira for analysis of CE-MS
and Yumino Kimura for technical assistance.
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Aple02000835: Dipeptide formation was confirmed in
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Senoo, Akihiro
