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The Sequential Action of a Dipeptidase and a

-Lyase Is
Required for the Release of the Human Body Odorant
3-Methyl-3-sulfanylhexan-1-ol from a Secreted
Cys-Gly-(S) Conjugate by Corynebacteria
*
□
S
Received for publication, January 28, 2008, and in revised form, May 9, 2008 Published, JBC Papers in Press, May 30, 2008, DOI 10.1074/jbc.M800730200
Roger Emter and Andreas Natsch
1
From the Bioscience Departement, Givaudan Schweiz AG, CH-8600 Duebendorf, Switzerland
Human axillary odor is formed by the action of Corynebacte-
ria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-
sulfanylhexan-1-ol in particular, form one key class of the odorif-
erous compounds. A conjugate with the dipeptide Cys-Gly has
been reported as the secreted precursor for 3-methyl-3-sulfanyl-
hexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the
major precursor of this odorant, with lower levels of the Cys-(S)
conjugate being present in axilla secretions. The enzymatic release
of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate
by the axilla isolate Corynebacterium Ax20 was thus investigated.
Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol
from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate,
whereas the previously isolated C-S lyase of this bacterial strain was
only able to cleave the Cys-(S) conjugate. o-Phenanthroline
blocked the release from the Cys-Gly-(S) conjugate but did not
affect cleavage of the Cys-(S) conjugate, indicating that in a first
step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond.
This enzyme was purified by four chromatographic steps and gel
electrophoresis, and the partial amino acid sequence was deter-
mined. The corresponding gene was cloned and expressed in Esch-
erichia coli. It codes for a novel dipeptidase with a high affinity
toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-
ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or
fresh axilla secretions with both the C-S lyase and the novel dipep-
tidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the
sequential action of these two enzymes from the skin bacterium
Corynebacterium Ax20 does release the odorant from the key
secreted precursor.
The skin in human armpits contains a dense arrangement of
sweat glands. Volatile substances evaporating from these areas
make a key contribution to human body odor. However, sweat
secreted from apocrine glands in these skin areas is initially
odorless, and since the work of Shelley et al. (1), it is known that
skin bacteria release the odoriferous principles from non-
smelling substrates present in these secretions. Indeed, the
axilla is a skin region colonized by an unusually dense bacterial
population, with a species composition dominated by the two
genera Staphylococcus and Corynebacterium (2, 3). Most indi-
viduals carry a flora that is dominated by either one of these two
genera, and there is a strong correlation between a high popu-
lation of Corynebacteria and strong axillary odor formation (2,
4). Subjects whose axillary skin is mainly colonized by Staphy-
lococci emit only low levels of odor. Based on this fundamental
work, axilla secretions contain non-odoriferous precursors that
are transformed into the volatile substances by bacterial
enzymes mainly present in Corynebacteria and to a lesser
extent in Staphylococci.
Early studies on the chemistry of human axilla odors identi-
fied the odoriferous steroids 5
␣
-androst-16-en-3-one (5, 6) and
5
␣
-androst-16-en-3
␣
-ol (7) in human axilla secretions. Later,
Zeng et al. (8) reported that short, branched fatty acids make a
major contribution to the axilla odor with (E)-3-methyl-2-hex-
enoic acid being the key component. In our previous work, we
had shown that a broad diversity of other unsaturated or
hydroxylated odorant acids is present in hydrolyzed axilla
secretions and that all these acids are released from the glands
in the form of odorless glutamine conjugates (9, 10). We had
isolated a specific zinc-dependent aminoacylase from the axilla
isolate Corynebacterium striatum Ax20, which catalyzes the
release of the odoriferous principles from these glutamine con-
jugates (9).
The third and most recently discovered class of human axilla
odorants are volatile sulfanylalkanols (11–13), with 3-methyl-
3-sulfanylhexan-1-ol (3M3SH)
2
as the quantitatively dominat-
ing compound within this structural class. This compound can
be released both from a Cys-(S) conjugate and from axilla secre-
tions by a C-S lyase cloned from Corynebacterium Ax20, indi-
cating that Cys-(S) conjugates could be physiological precur-
sors for this compound class (12). However, it was later shown
that a Cys-Gly-(S) conjugate of 3M3SH is secreted by human
subjects (14) (for structures, see Fig. 1) and that bacterial cul-
tures of Staphylococcus haemolyticus can release 3M3SH from
* The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the Gen-
Bank
TM
/EBI Data Bank with accession number(s) EU311559.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
three supplemental figures.
1
To whom correspondence should be addressed: Ueberlandstrasse 138,
CH-8600 Duebendorf, Tel.: 41-44-8242105; Fax: 41-44-8242926; E-mail:
andreas.natsch@givaudan.com.
2
The abbreviations used are: 3M3SH, 3-methyl-3-sulfanylhexan-1-ol;
GC-FPD, gas-chromatography with flame photometric detection; LC, liq-
uid chromatography; MS, mass spectrometry; MS
2
, two-stage mass analy
-
sis; HPLC, high pressure liquid chromatography; Ni-NTA, nickel-nitrilotri-
acetic acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 30, pp. 20645–20652, July 25, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
JULY 25, 2008 • VOLUME 283 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20645
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this dipeptide precursor. In a recent patent application, this
activity was attributed to a

-lyase, but the corresponding
enzyme was neither isolated from S. haemolyticus, nor has it
been characterized (15). Thus, the enzymatic release of the key
axilla odorant 3M3SH by skin bacteria from the physiological
dipeptide precursor has not yet been deciphered. Here, we
report the isolation and the characterization of a novel specific
dipeptidase and the corresponding gene from the axilla isolate
Corynebacterium Ax20. We show that the secreted Cys-Gly-(S)
conjugate of 3M3SH first needs to be cleaved by this dipepti-
dase and only afterward becomes a substrate of the previously
reported C-S lyase, which finally releases 3M3SH.
EXPERIMENTAL PROCEDURES
Materials—Unless otherwise noted, all chemicals were pur-
chased from Fluka (Buchs, Switzerland). (Z)-protected amino
acids and peptides as enzyme substrates were from Aldrich
(Buchs, Switzerland) and from Senn Chemicals (Dielsdorf,
Switzerland). All columns and chromatography resins were
from Amersham Biosciences (Otelfingen, Switzerland) with the
exception of Ni-NTA agarose purchased from Qiagen (Hom-
brechtikon, Switzerland). (S)-(1-(2-hydroxyethyl)-1-methylbu-
tyl)-
L-cysteinylglycine (Cys-Gly-(S) conjugate) was synthesized
by the method described by Starkenmann et al. (14). 3M3SH, its
Cys-(S) conjugate, and Gln conjugates of carboxylic acids as
reference substrates were synthesized as described before (9,
10, 12). The recombinant C-S lyase from Corynebacterium
Ax20 was expressed and purified on a Ni-NTA-affinity column
(12). Axilla secretions of individual donors were sampled on
cotton pads fixed in the axillary region during physical exercise
(10).
Bacterial Strains—Isolation and characterization of axilla
bacteria were described previously (9). The bacterial strains
were grown on tryptic soy agar supplemented with 0.01%
Tween 80 as lipid source. For enzyme purification or enzyme
assays, axilla bacteria were grown in Mueller-Hinton broth sup-
plemented with 0.01% Tween 80. E. coli strain TOP10, used for
the expression of recombinant enzymes, was grown in LB
broth.
LC-MS Analysis of Axilla Secretions—The aqueous fraction
of axilla secretions was fractionated over a Superdex peptide
10/300 GL column using (NH
4
)
2
CO
3
(100 mm) as elution
buffer, and the single fractions were
analyzed with LC-MS with the
method laid out in Natsch et al. (10).
In brief, a Finnigan LCQ mass spec-
trometer operated in the atmo-
spheric pressure chemical ioniza-
tion mass spectrometry mode and
equipped with a Flux Rheos 2000
HPLC pump was used, and HPLC
separation was performed on a C18
RP column modified for proteins
and peptides (Grace Vydac, Hespe-
ria, CA). The mobile phase con-
sisted of H
2
O (A) and MeOH (B)
each containing 1% HOAc (v/v).
GC-FPD Analysis for Release of
3M3SH—The Cys- and Cys-Gly-(S) conjugates of 3M3SH or
the aqueous fraction of axilla secretions were incubated with
the recombinant enzymes or bacterial extracts for 2 h. The
aqueous phase (500
l) was extracted with 250
l of methyl-
tert-butyl-ether, and 6
l were injected in the splitless pulse-
pressure mode onto a SPW1-sulfur column (Supelco, Belle-
fonte, PA) mounted on an Agilent GC 6890N (Agilent,
Wilmington, DE) system with a flame photometric detector
specific for sulfur chemicals. The temperature program was set
to 2 min of initial temperature at 50 °C, heating at a rate of
10 °C/min to 240 °C, and a final 15 min at 240 °C.
Dipeptidase Activity Assay—Bacterial extracts, column frac-
tions, or the purified peptidase were incubated with the Cys-
Gly-(S) conjugate or (S)-benzyl-Cys-Gly at a final concentra-
tion of 0.5 or 1 m
M and with an excess of

-lyase (10
g/ml) in
buffer A (50 m
M NaCl, 50 mM NaH
2
PO
4
/K
2
HPO
4
,pH7)ina
final volume of 100
l. Release of 3M3SH or benzylthiol was
detected by adding 50
lofa1mM solution of the thiol-specific
fluorescent probe monobromobimane dissolved in NaCO
3
buffer (100 mM, pH 8.8) and, after a 5-min reaction time, fluo-
rescence measurement on a Flexstation (Molecular Devices,
Sunnyvale, CA) with an excitation at 390 nm and emission at
478 nm. To determine enzyme kinetics, the same assay was
used, but the enzyme reaction with the dipeptidase was stopped
by adding EDTA (0.1 m
M), and only then the

-lyase was added
to release the thiol for fluorescent detection.
Thin Layer Chromatography for Peptide Cleavage—Cys- and
Cys-Gly-(S) conjugates, dipeptides and acetyl ornithine (1 m
M
solutions) were incubated with the peptidase for 1 h. 10
lof
each reaction were spotted on TLC plates and developed with a
mixture of 1-butanol, acetic acid, H
2
O (4:1:1) in the case of the
Cys- and Cys-Gly-(S) conjugates or with H
2
O and 1-propanol
(1:1) in the case of the dipeptides. Products were visualized by
spraying a ninhydrin solution and heating the plates until the
completion of the ninhydrin reaction.
Protein Determination and SDS-PAGE—Protein concentra-
tions were determined with the Bradford reagent (Bio-Rad)
using bovine serum albumin as standard. SDS-PAGE was per-
formed according to Laemmli (16) with 5% stacking gels and
10% separation gels. Protein bands were visualized by Coomas-
sie Blue staining.
FIGURE 1. Proposed scheme for the release of 3M3SH from the Cys-Gly-(S) conjugate.
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Purification of the Peptidase—C. striatum Ax20 was selected
for the purification of the enzyme responsible for the cleavage
of the Cys-Gly-(S) conjugate. It was grown for 42 h, and cells
from a total culture volume of four liters were washed once with
buffer A and resuspended in a final volume of 5 ml of buffer A.
Using glass beads (425– 600
m, Sigma), the cells were
mechanically disrupted by vortexing them at maximal speed for
20 min. The lysate was cleared by centrifugation and passed
through a 0.45-
M syringe filter. The extract was then sequen-
tially run over four chromatography columns: 1) phenyl-sepha-
rose hydrophobic interaction resin, elution with a linear gradi-
ent from 1000 to 0 m
M (NH
4
)
2
SO
4
in Buffer A; 2) Mono Q
strong anion exchange column on the fast protein liquid chro-
matography system, elution with a gradient from 0 to 800 m
M
KCl in buffer A; 3) Mono P weak anion exchange column on the
fast protein liquid chromatography system, elution with a gra-
dient from 0 to 800 m
M KCl in 50 mM bis-Tris buffer (pH 6.5); 4)
Superdex 200 gel filtration column, elution with buffer A. After
each column separation, active fractions (determined by the
fluorescent assay) were pooled and desalted by dilution/ultra-
filtration. Fractions of the final Superdex gel filtration step were
run on a 10% SDS-PAGE gel. The single protein band common
to the active fractions was isolated and subjected to tryptic
digestion, and the sequence of internal peptides was deter-
mined with LC-electrospray mass ionization-tandem MS anal-
ysis (Genomic Center, University of Zu¨rich, Switzerland).
Molecular Biology Methods—Chromosomal DNA of Ax20
was obtained from cell lysates by proteinase K digestion and
subsequent extraction with cetyltrimethylammonium bro-
mide/NaCl and chloroform/isoamylacetate (17). Based on the
partial amino acid sequences of the purified enzyme, degener-
ated primers were designed to amplify genomic DNA frag-
ments. Standard PCR conditions were used according to the
manufacturer (Taq polymerase, Sigma, Buchs, Switzerland).
The amplified DNA was submitted to Microsynth (Balgach,
Switzerland) for sequence analysis. Based on the obtained par-
tial sequence, specific nested oligonucleotides were designed to
clone the upstream and downstream regions. Chromosomal
DNA of Ax20 was digested with SmaI and PvuII and ligated to
the GenomeWalker Adaptor (Clontech). The upstream and
downstream regions were then amplified as described in the
instructions to the Universal GenomeWalker
TM
kit (Clontech
Laboratories) and sequenced. The resulting open reading frame
was amplified from chromosomal DNA of Ax20 by PCR using
the specific primers 5⬘-CGA CAT GCC ATG GGC AGC AAC
GAC AAG GCA GCA ACC AGC-3⬘ and 5⬘-CGA CAT AAG
CTT TTT CCC GTA GGT GAG CAG GAA T-3⬘. The ampli-
fied DNA fragment was digested with NcoI and HindIII and
ligated into the vector pBAD/myc-HisA (Invitrogen, Gronin-
gen, The Netherlands) predigested with the same enzymes. The
resulting plasmid pBAD/mycHis-tpdA was transformed into
the host strain E. coli TOP10 (Invitrogen). This strain was
grown in LB broth until it reached an A
600
of 0.5. The culture
was supplemented with arabinose (0.2% final concentration) to
induce gene expression, further incubated for 4 h, and har-
vested by centrifugation, and the cells were disrupted using a
French Press. The His-tagged recombinant peptidase was
finally purified using a Ni-NTA column according to the man-
ufacturer’s instructions.
RESULTS
LC-MS Analysis of Axilla Fractions to Detect Amino Acid
Conjugates of 3M3SH—Aqueous fractions of axilla secretions
obtained by gel filtration were analyzed by LC-MS in compari-
son with synthetic Cys- and Cys-Gly-(S) conjugates of 3M3SH.
In axilla secretions, a significant peak at retention time 4.85 min
was observed on the extracted mass trace m/z 293, correspond-
ing to the protonated Cys-Gly-(S) conjugate (Fig. 2, A-2 and
B-2). This peak can only be detected in axilla secretions on the
selected ion trace, whereas the total ion current (TIC) trace
contains two dominant peaks (Fig. 2, B-1), which correspond to
the two key Gln conjugates of carboxylic acids reported before
(9), as verified based on MS analysis and comparison with
synthetic references (data not shown). The Cys-(S) conju-
gate of 3M3SH yields an 8-fold lower signal intensity in
LC-MS analysis as compared with the Cys-Gly-(S) conjugate
(compare the different normalization level NL in Fig. 2, A-2
and A-3), probably due to less efficient ionization of the Cys-
(S) conjugate. In axilla secretions, on the m/z trace 236, only
a very minor peak at the correct retention time was observed,
putatively corresponding to the Cys-(S) conjugate (indicated
in Fig. 2, B-3 by an arrow). Indeed, the on-line MS
2
spectrum
of this peak showed the same base ion at m/z 122 as the MS
2
spectrum of the synthetic reference sample. This ion pre-
sumably corresponds to the protonated free Cys (supple-
mental Fig. S1). Nevertheless, even considering the lower
response factor for the Cys-(S) conjugate, this compound,
albeit clearly present, is the minor component, and thus, the
Cys-Gly-(S) conjugate of 3M3SH indeed is the main precur-
sor present in axilla secretions.
3M3SH Is Released from the Cys-Gly-(S) Conjugate by the
Sequential Action of a Metallopeptidase and a

-Lyase—The
fact that mainly the Cys-Gly-(S) conjugate and only small
amounts of the Cys-(S) conjugate can be found in fresh sweat
suggests that 3M3SH is directly released either by a

-lyase in
axilla bacteria from the Cys-Gly-(S) conjugate or from a Cys-( S)
conjugate that has been generated from the Cys-Gly-(S) conju-
gate by hydrolysis of the Cys-Gly peptide bond. To test the two
hypotheses, the synthetic Cys- and the Cys-Gly-(S) conjugates
of 3M3SH were incubated with the purified

-lyase from the
axilla strain Ax20 or with total cell extracts of several bacterial
strains isolated from the axilla. As shown in Fig. 3, the purified

-lyase and the Ax20 extract released significant amounts of
3M3SH from the Cys-(S) conjugate, and this could not be
blocked by the metallopeptidase inhibitor o-phenanthroline.
When incubated with the Cys-Gly-(S) conjugate, only the Ax20
extract but not the recombinant

-lyase released a significant
amount of 3M3SH. Interestingly, this reaction could be blocked
by o-phenanthroline, suggesting that the strain Ax20 harbors
an o-phenanthroline-sensitive dipeptidase that hydrolyzes the
Cys-Gly-(S) conjugate, thereby producing the substrate for the

-lyase. Extracts of Staphylococcus and Micrococcus strains did
not cleave either of the two conjugates, whereas other tested
Corynebacteria showed only weak

-lyase activity (Fig. 3).
Corynebacterium jeikeium K411 is the only axilla isolate whose
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genome had been sequenced (19). After a 2-h incubation, no
significant cleavage of the Cys-Gly-(S) conjugate by this strain
was observed; however, after prolonged incubation it slowly
cleaved this substrate (data not shown). The structures of the
conjugates along with these proposed reactions are shown in
Fig. 1.
Purification of the Novel Metallopeptidase and Cloning of the
Corresponding Gene—The unknown metallopeptidase cleaving
the Cys-Gly-(S) conjugates of 3M3SH was then purified from
cellular extracts of C. striatum Ax20
by activity-guided fractionation as
described under “Experimental Pro-
cedures.” Two columns were tested
for the first purification step, a
Mono-Q and a phenyl-Sepharose
column. From both columns, the
metallopeptidase activity eluted as a
single peak. Also, from all subse-
quent column fractionations, only a
single peak of activity was recov-
ered, indicating that only one key
enzyme is involved in the hydrolysis
of the Cys-Gly-(S) conjugate. Frac-
tions of the last purification step
were analyzed by SDS-PAGE. Three
different polypeptides were left in
the active fractions. Comparison
of the relative peptidase activity
and the intensity of the bands
resulted in a clear candidate pro-
tein, which had an apparent mass
of ⬃50 kDa (see supplemental Fig.
S2 in the supporting information).
The candidate protein was excised
and submitted to a tryptic digest
and sequence analysis, leading to
the amino acid sequence of several
internal peptides.
A data base search with the
obtained peptide sequences revealed
homology to putative peptidases,
suggesting that the analyzed protein
could indeed be responsible for the
cleavage of the Cys-Gly bond. Based
on four peptide sequences, degener-
ated primers were designed, and a
total of 20 primer combinations
were used for PCR amplification
with chromosomal DNA of Ax20 as
template. Two primer combina-
tions led to specific products of 281
and 272 bp, respectively. The
obtained PCR products were
sequenced, and based on these
partial sequences, oligonucleo-
tides were designed to clone the
upstream and downstream regions
by genome walking using libraries
generated from PvuII and SmaI digests of chromosomal
Ax20 DNA. An upstream fragment of 600 bp and a down-
stream fragment of 2400 bp were obtained. Within these
sequenced regions, open reading frames coding for the
N-terminal part and the C-terminal part were identified.
Finally, the complete open reading frame was amplified by
PCR using Ax20 genomic DNA as template, and it was
cloned into the bacterial expression vector pBAD/mycHisA.
The sequence was deposited in the GenBank
TM
under acces
-
FIGURE 2. LC-atmospheric pressure chemical ionization (ⴙ)-MS analysis of synthetic Cys- and Cys-Gly-(S)
conjugates of 3M3SH (A) and unhydrolyzed axilla secretions fractionated by gel filtration (B). A, a solu-
tion containing each 100
M of the Cys- and Cys-Gly-(S) conjugates of 3M3SH. B, the fraction eluting after 20.5
ml from a Superdex peptide 10/300 GL column loaded with pooled axilla secretions from two donors. A-1 and
B-1, total ion current (TIC) chromatograms (mass range m/z 80 –600); A-2 and B-2, extracted mass chromato-
grams of m/z 293 ([M⫹H]
⫹
of the Cys-Gly-(S) conjugate); A-3 and B-3, extracted mass chromatograms of m/z
236 ([M⫹H]
⫹
of the Cys-(S) conjugate). The largest peak in each chromatogram is normalized to 100, and the
normalization level NL is indicated in the graphs.
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sion number EU311559. The gene was named tpdA, which
stands for “thiol precursor dipeptidase.”
Sequence Comparison with Related Proteins—The protein
deduced from the open reading frame has a high homology to a
large number of putative bacterial peptidases belonging to the
M20 family of metallopeptidases. Closely related genes, for
which no function has been identified yet, exist in the genomes
of most other members of the class of actinobacteria. The clos-
est homologues in E. coli are the succinyl-diaminopimelate des-
uccinylase and the acetyl-ornithine deacetylase (18), two
related proteins also belonging to
the M20 family of peptidases, which
are involved in the biosynthesis of
lysine and arginine, respectively.
The sequence alignment of the
dipeptidase with these genes of
known function, with the closest
relatives from Corynebacterium
diphteriae and C. jeikeium K411
(19) and with the carboxypepti-
dase G2 from Pseudomonas
aeruginosa (18, 20), is shown in
supplemental Fig. S3 in the sup-
porting information.
Characterization of the Pure
Recombinant Enzyme—Transfor-
mants of E. coli strain TOP10 har-
boring the plasmid pBAD/mycHis-
tpdA expressed high levels of the
His-tagged recombinant protein, which was purified to ⬎95%
purity using a Ni-NTA column. To demonstrate that the iso-
lated protein indeed catalyzes the hydrolysis of the peptide
bond of the Cys-Gly-(S) conjugate, this substrate was incubated
with the purified protein, and the reaction products were sep-
arated by TLC. As shown in Fig. 4, the Cys-Gly-(S) conjugate
was indeed hydrolyzed to the Cys-(S) conjugate and glycine,
and the same reaction was performed by the TpdA with (S)-
benzyl-Cys-Gly as a substrate (data not shown).
Having shown this, we tried to find additional substrates for
the identified enzyme. A series of 24 dipeptides was incubated
for 1 h with the peptidase and then analyzed by TLC. The pep-
tidase did hydrolyze a relatively broad range of dipeptides, but it
was not able to cleave acidic amino acids from the C terminus
(Table 1). In addition, the peptidase did not hydrolyze dipep-
tides with glycine in the second position unless a bulky hydro-
phobic residue was present in the N-terminal position. Thus,
although (S)-substituted Cys-Gly conjugates are very good sub-
strates, unsubstituted Cys-Gly is not cleaved.
To verify that the recombinant enzyme indeed has the same
substrate specificity as the native enzyme, the purified fraction
after the last purification step (Superdex gel filtration) was
incubated with 13 of the potential substrates, and the substrate
specificity was compared with the recombinant enzyme. As
illustrated in Table 1, column 3, the specificity of both prepara-
tions is essentially the same.
As mentioned above, the closest homologues in E. coli are the
succinyl-diaminopimelate desuccinylase and the acetyl-orni-
thine deacetylase. To test whether the isolated peptidase also
harbors deacylase activity, it was incubated with acetyl-
L-orni-
thine, but no deacylation was detected. N
␣
-Acyl-Gln conju
-
gates for odorant acids are secreted in the axilla and are
substrates of the recently identified enzyme N
␣
-acyl-Gln-ami
-
noacylase also belonging to the M20 class of peptidases (9).
Since two dipeptides with Gln in second positions served as
substrates for the dipeptidase, we tested whether the N
␣
-acyl-
Gln conjugates can also serve as substrates for TpdA. Interest-
ingly, we found that these glutamine conjugates are indeed
hydrolyzed by the peptidase to a certain extent (Table 1).
FIGURE 3. Release of 3M3SH from synthetic conjugates by bacterial extracts and recombinant

-lyase.
Total cell extracts (0.25 mg of protein/ml) of bacterial strains isolated from axillary skin and

-lyase (0.005
mg/ml) were incubated with the Cys- or the Cys-Gly-(S) conjugates (0.5 mM) of 3M3SH for 2 h. Where indicated,
o-phenanthroline was added at a final concentration of 0.5 mM. Released 3M3SH was detected using the
fluorescent dye monobromobimane. Species assignment is as follows: Ax1, Staphylococcus capitis; Ax6, Staph-
ylococcus epidermidis; Ax9, Micrococcus luteus; Ax15, C. jeikeium; Ax19, C. jeikeium; Ax20, C. striatum; Ax21,
Corynebacterium bovis; and K411, C. jeikeium.
FIGURE 4. Cleavage of the Cys-Gly-(S) conjugate by TpdA. The Cys-Gly-(S)
conjugate of 3M3SH (1 mM) was incubated with increasing amounts of the
TpdA for 1 h. 10
l of each reaction were spotted on a TLC plate and devel-
oped with 1-butanol:acetic acid:H
2
O (4:1:1).
A Biochemical Mechanism of Human Axillary Odor Formation
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The Cys-Gly-(S) conjugates of 3M3SH and benzylthiol were
finally used to determine enzyme kinetics. The non-physiolog-
ical benzylthiol conjugate was chosen as an alternative sub-
strate for analysis of enzyme activity as it is the simplest Cys-Gly
conjugate available. A K
m
value of 0.045 mM and a V
max
of 0.023
mmol䡠min
⫺1
䡠mg
⫺1
was obtained for the hydrolysis of the Cys-
Gly-(S) conjugate of 3M3SH. For the hydrolysis of the Cys-Gly-
(S) conjugate of benzylthiol, we determined a K
m
value of 0.20
m
M and a V
max
of 0.19 mmol䡠min
⫺1
䡠mg
⫺1
(data not shown).
These data correlate with the results listed in Table 1, suggest-
ing that the dipeptidase cleaves the benzylthiol precursor more
efficiently at high substrate concentrations. At low substrate
concentrations, as found in the secreted axilla sweat, the dipep-
tidase shows a significantly higher affinity for the physiological
Cys-Gly-(S) conjugate of 3M3SH.
The homologous gene jk0266 from Corynebacterium
jeikeium K411 (19) was also cloned and expressed. It cleaves the
Cys-Gly-(S) conjugate of benzylthiol with a K
m
value of 0.28 mM
andav
max
of 0.16 mmol䡠min
⫺1
䡠mg
⫺1
, but the Cys-Gly-(S) con
-
jugate of 3M3SH was cleaved with a 25-fold lower v
max
by this
related enzyme as compared with TpdA. Thus, not all Coryne-
bacterium strains isolated from the axillary skin have an
enzyme perfectly adapted to this substrate (data not shown).
The recombinant TpdA was incubated with the metal che-
lating agents o-phenanthroline and pyridine-2,6-dicarboxylic
acid. The IC
50
of both these compounds is 15
M. This confirms
the conclusion from the results in Fig. 3, namely that the
responsible enzyme for the cleavage of the Cys-Gly conjugates
is a o-phenanthroline sensitive metallopeptidase.
Release of 3M3SH from Axilla Secretions by the Recombi-
nant Enzymes—To finally prove the involvement of the novel
dipeptidase in the release of 3M3SH from axilla secretions, a
pooled axilla secretion sample from two donors was split
into four equal portions and left untreated (A) or treated
with the dipeptidase (B), the

-lyase (C), both the dipepti-
dase and the

-lyase (D). The samples were analyzed by GC-
FPD, and the resulting chromatograms as compared with syn-
thetic 3M3SH. The untreated and the dipeptidase-treated samples
contained no 3M3SH, whereas the

-lyase did only release a small
quantity of 3M3SH (0.5
g/ml; indicated in Fig. 5C by an arrow).
The combined action of both enzymes released a 14-fold higher
FIGURE 5. GC-FPD analysis of axilla secretions treated with the TpdA and
the

-lyase. The aqueous fraction from axilla secretions pooled from two
donors was split into four portions, treated with the enzymes (10
g/ml) for
2 h, extracted with solvent, and analyzed with GC with a sulfur specific detec-
tor. A, untreated sample; B, sample treated with the dipeptidase TpdA;
C, sample treated with the

-lyase; D, sample treated with TpdA and

-lyase; and E, 2 ppm of synthetic 3M3SH as reference.
TABLE 1
Substrate specificity of TpdA and the native enzyme preparation
Substrate
Recombinant
TpdA
Native
enzyme
preparation
b
Dipeptides and derivatives
(S) 3M3SH-Cys-Gly ⫹⫹ ⫹⫹
(S) Benzyl-Cys-Gly ⫹⫹⫹ ⫹⫹⫹
Cys-Gly ⫺⫺
Gly-Gly ⫺ ND
Lys-Gly ⫺ ND
Phe-Gly ⫹⫹ ⫹⫹
Trp-Leu ⫹⫹⫹ ⫹⫹⫹
Glu-Trp ⫹⫹⫹ ⫹⫹
Ile-Asn ⫹⫹⫹ ND
Leu-Ala ⫹⫹⫹ ND
Leu-Asn ⫹⫹⫹ ND
Val-Lys ⫹⫹⫹ ND
Ala-Ala ⫹⫹ ⫹⫹
Ser-Leu ⫹⫹ ND
Thr-Gln ⫹⫹ ND
Val-Gln ⫹⫹ ND
Ile-Leu ⫹⫹
Trp-Val ⫹ ND
Ala-Glu ⫺⫺
Arg-Glu ⫺ ND
Asp-Asp ⫺⫺
Asp-Glu ⫺ ND
Glu-Asp ⫺ ND
Glu-Glu ⫺⫺
Ile-Trp ⫺⫺
Ile-Val ⫺ ND
Val-Glu ⫺ ND
Acyl-amino acids ND
Acetyl-ornithine ⫺ ND
N
␣
-Lauroyl-Gln
⫹ ND
N
␣
-(E)-3-methyl-2-hexenoyl-glutamine
⫹/⫺ ND
N
␣
-3-methyl-3-hydroxy-hexanoyl-glutamine
⫹/⫺ ND
a
1mM substrate was incubated with different concentrations of TpdA for 1 h. ⫺
indicates no hydrolysis; ⫹/⫺ indicates traces of product detected with 1
g/ml
enzyme; ⬎20% of substrate hydrolyzed with 1
g/ml enzyme; ⫹⫹ indicates ⬎20%
of substrate hydrolyzed with 0.3
g/ml enzyme; ⫹⫹⫹ indicates ⬎20% of sub-
strate hydrolyzed with 0.1
g/ml enzyme; ⫹⫹⫹⫹ indicates ⬎20% of substrate
hydrolyzed with 0.03
g/ml enzyme.
b
1mM substrate was incubated with different dilutions of the native enzymatic
preparation obtained after the fourth column (Superdex gel filtration). ⫺ indicates
no hydrolysis; ⫹/⫺ indicates traces of product detected with 1:1 dilution; ⫹ indi-
cates ⬎20% of substrate hydrolyzed with 1:1 dilution; ⫹⫹ indicates ⬎20% of
substrate hydrolyzed with 1:3 dilution; ⫹⫹⫹ indicates ⬎20% of substrate hydro-
lyzed with 1:10 dilution; ⫹⫹⫹⫹ indicates ⬎20% of substrate hydrolyzed with 1:30
dilution. The native preparation used had an apparent purity of 30%. ND, not
determined.
A Biochemical Mechanism of Human Axillary Odor Formation
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quantity of 3M3SH as compared with the incubation with the

-lyase only (7
g/ml; Fig. 5D). In addition, several further small
peaks for sulfur-containing compounds were formed by the com-
bined enzyme treatment, indicating that further sulfur volatiles
could be present in axilla secretions as Cys-Gly-(S) conjugates and
are released by the same two enzymes.
DISCUSSION
Based on the fact that a C-S lyase from Corynebacterium
Ax20 can release 3M3SH both from a synthetic Cys-(S) conju-
gate and from axilla secretions, we had proposed that a Cys-(S)
conjugate might be the key secreted precursor (12). However,
the conjugate with the dipeptide Cys-Gly was later reported as
the key precursor for 3M3SH (14). The presented LC-MS data
now confirm the Cys-Gly-(S) conjugate as the major precursor
of 3M3SH, but, in combination with the LC-MS
2
analysis, they
clearly show that the Cys-(S) conjugate is also present, albeit at
lower levels. This reminds of the biochemical precursors for the
key odorant 3-methyl-3-sulfanyl-butan-1-ol in cat urine. Cat
urine contains as a precursor the unusual amino acid felinine
((S)-(1,1-dimethyl-3-hydroxypropyl)-cysteine (21)), and it was
later shown that in addition to the Cys-(S) conjugate felinine,
cat urine also contains the corresponding Cys-Gly-(S) conju-
gate (22). Interestingly, in cats, unlike in most other mammals,
urine contains a higher level of protein, and it was shown that
this protein level is mainly due to a single protein named Cauxin
(carboxylesterase-like urinary excreted protein (23)), which
cleaves the Cys-Gly-(S) conjugate and releases felinine (24).
Therefore the cleavage of this precursor is performed by a car-
boxypeptidase that is physiologically secreted in the cat.
Here, we show that on the human skin, the cleavage of the
Cys-Gly-(S) conjugate can be performed by skin commensal
microorganisms rather than by a secreted human enzyme. The
novel dipeptidase reported from the skin isolate Corynebacte-
rium Ax20 has a very high affinity to the secreted Cys-Gly-(S)
conjugate, but it also cleaves a range of dipeptides. The fact that
the dipeptidase has a low K
m
for the Cys-Gly-(S) conjugate
could indicate that the skin bacteria have adapted their enzyme
specifically for this type of substrate present in the natural hab-
itat of this bacterial strain. On the other hand, the V
max
for the
physiological substrate is relatively low. It is noteworthy that
even if the enzymatic cleavage is relatively slow, it may quickly
yield a perceivable quantity of the odorant since the human
nose is very sensitive to 3M3SH. We had measured the sensory
threshold (12) to be 1 pg/liter air, indicating that 0.007 nmol is
perceivable in 1 m
3
air.
We could detect cleavage of the Cys-Gly-(S) conjugate only
in strains of Corynebacteria, which is in agreement with the well
known fact that human subjects with a high population of
Corynebacteria can form axillary malodor (2, 25). However,
Starkenmann et al. (14) have reported that an isolate of
S. haemeolyticus is releasing 3M3SH from the Cys-Gly-(S) con-
jugate. Thus, it appears that members of different bacterial gen-
era present on axillary skin have adopted their enzymes to these
secreted substrates.
Based on homology searches, the novel thiol precursor
dipeptidase TpdA belongs to the M20 class of peptidases. Most
bacterial species sequenced in recent years do contain a homo-
logue of this gene. However, no function has been associated to
the 75 most closely related sequences investigated. The closest
relatives with a known function are the two enzymes DapE and
ArgE involved in the biosynthesis of lysine and arginine, respec-
tively, which were characterized in E. coli and other bacteria
(18, 26, 27), and the carboxypeptidase G2 form P. aeriginosa.
Interestingly, among the five residues involved in zinc binding
in carboxypeptidase G2 (28), four are also conserved in TpdA.
The final proof that both enzymes, the novel thiol precursor
dipeptidase and the

-lyase, are needed to cleave the Cys-Gly-(S)
conjugate comes from co-incubations of either the synthetic com-
pound or the axilla secretions with both enzymes. From axilla
secretions, the

-lyase alone can release low quantities of 3M3SH,
as reported earlier (12), which is in agreement with the LC-MS
finding that low levels of the Cys-(S) conjugate are present in these
secretions; however, much larger quantities of 3M3SH are
released by the joint action of the

-lyase and the dipeptidase.
Interestingly, the dipeptidase also has a certain affinity
toward the N
␣
-acyl-Gln conjugates of odorant acids in the axilla
reported before (9). On the other hand, the previously isolated
enzyme N
␣
-acyl-Gln-aminoacylase, which also belongs to the
M20 class of peptidases and which is specific for these Gln
conjugates, has a very low but detectable catalytic activity cleav-
ing the Cys-Gly-(S) conjugate (data not shown). Thus, the two
related metallopeptidases involved in the formation of these
very different structural classes of key axilla odorants have a
certain cross-specificity. In the pharmaceutical field, it was
shown that dual metallopeptidase inhibitors can be developed,
e.g. for the two metallopeptidases angiotensin converting
enzyme and neutral endopeptidase, which both are involved in
neuropeptide processing and regulation of blood pressure (29).
Since two metallopeptidases with a certain cross-specificity
both are involved at key stages of axilla odor formation, the
medicinal chemistry approach of designing dual metallopepti-
dase inhibitors could, in the future, be applied to develop next
generation ingredients for cosmetic deodorants.
The dipeptidase identified in this work and the previously
identified N
␣
-acyl-Gln-aminoacylase and

-lyase enzymes are
all involved in the release of odorants from precursors. This
release is time-dependent, and thus, fresh sweat secreted dur-
ing physical exercise initially is odorless. It is a common obser-
vation that under certain conditions of nervous tension (and
thus, maybe under a specific hormonal regulation), an immedi-
ately perceivable body odor is formed. This very particular odor
might be directly secreted without bacterial/enzymatic action
on the axilla secretions being necessary. The chemical nature
and the origin of this specific odor cannot yet be explained by
the detailed analytical and enzymatic studies presented here or
published previously, and thus, forms a potential subject of fur-
ther research.
Acknowledgments—We give our thanks to F. Flachsmann for synthe-
sizing reference compounds, H. Gfeller for LC-MS analysis, B. Schill-
ing for critical discussions, and Dr. Peter Hunziker (Functional
Genomics Center Zurich, Zu¨rich, Switzerland) for amino acid
sequence analysis.
A Biochemical Mechanism of Human Axillary Odor Formation
JULY 25, 2008 • VOLUME 283 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 20651
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A Biochemical Mechanism of Human Axillary Odor Formation
20652 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283• NUMBER 30 • JULY 25, 2008
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SUPPLEMENTAL INFORMATION, Emter & Natsch
Figure S1
Figure S1. LC-APCI(+)-MS and MS
2
analysis of (A) synthetic Cys-(S)-conjugate of 3M3SH and (B)
unhydrolyzed axilla secretions fractionated by gel filtration. A, a solution containing 100 μM of the
Cys-(S)-conjugates of 3M3SH; and B, the fraction eluting after 20.5 ml from a Superdex Peptide
10/300 GL column loaded with pooled axilla secretions from two donors. A-1 and B-1, selected ion
monitoring (SIM) chromatograms of m/z 236 ([M+H]
+
of the Cys-(S)-conjugate); A-2 and B-2,
selected reaction monitoring (SRM) chromatograms (fragmentation of the precursor ion m/z 236 to
the product ion m/z 122); A-3 and B-3, product ion spectra (MS
2
) of m/z 236 at retention time 4.82.
Figure S2, supporting information.
0
1000000
2000000
3000000
4000000
5000000
Fr. 12
Fr. 13
Fr. 14
Fr. 15
Fr. 16
Fr. 17
Fluorescence
Figure S2. SDS-Page of the active fractions after the 4
th
purification step (A), and relative activity of
the different fractions 12 – 17 (B). Lane 1: Size marker; Lane 2: pooled fractions after the Mono Q
step; Lane 3: Pooled fractions after the Mono P step, Lane 4 – Lane 9: Fractions 12 – 17 after
Superdex 200 gel filtration.
12 13 14 15 16 17
A
B
66 kDa
45 kDa
31 kDa
C.stri_TpdA 1 ----------------------MSNDKAATSTNFNLTPNRERIFQELSELISHYSPHSMP
C.diph_DIP2037 1 -----------------------MTKTAIQTVRPLVEKQRERIFKDLSEITSYNSVHSTP
C.jeik_jk0266 1 ----------------MSDTNSESNTSQLDLARAAIAEQMPQLKEDLTTLVSFESVHSAP
P.aeru_Cpg2 1 MHARRLPRLLPLALAFLLSPAAFAADTPAAELLRQAEAERPAYLDTLRQLVAVDSGTGQA
E.coli_ArgE 1 -----------------------------------MKNKLPPFIEIYRALIATPSISATE
E.coli_DapE 1 --------------------------------------MSCPVIELTQQLIRRPSLSPDD
C.stri_TpdA 39 EHADT-HEEAAKWVTAKLEELGLDVTRHPTVDDADTIIGVKEPVGDAPTILLYSHYDVVP
C.diph_DIP2037 38 ECAED-HAAACAWIVNALKEADLNVTEYLYDGGATTVIGTKEPEDGAPTVLLYCHYDVVP
C.jeik_jk0266 45 GLEEA-NAAAAQWVIDTFTSVGIPVEGHVTTDGSTSVIGLREPAEGYPTILLYSHFDVQP
P.aeru_Cpg2 61 EGLG----QLSALLAERLQALGAQVRSAPATPSAGDNLVATLDGTGSKRFLLMIHYDTVF
E.coli_ArgE 26 EALDQSNADLITLLADWFKDLGFNVEVQPVPGTRNKFNMLASIGQGAGGLLLAGHTDTVP
E.coli_DapE 23 AGCQA-------LLIERLQAIGFTVERMDFADTQNFWAWRGQ----GETLAFAGHTDVVP
His 112
C.stri_TpdA 98 AQNPAVWTNDPLELDERDGRWYGRGAADCKGNVIMHLEALRMVQEN------GGTDLGLK
C.diph_DIP2037 97 AGDPTAWESDPFTLTERNGRWYARGAADCKGNIAMHLAALRAVKEA------GGTKLGIK
C.jeik_jk0266 104 AGDIEAWTNDPWTLTERDGRWYGRGTADCKGHVAMHVAVLRALSILSDAHFPAAKNLGIR
P.aeru_Cpg2 117 AAG----SAAKRPFREDAERAYGPGVADAKGGVAMVLHALALLRQQG-----FRDYGRIT
E.coli_ArgE 86 FDD-GRWTRDPFTLTEHDGKLYGLGTADMKGFFAFILDALRDVDVT-------KLKKPLY
E.coli_DapE 72 PGDADRWINPPFEPTIRDGMLFGRGAADMKGSLAAMVVAAERFVAQHP-----NHTGRLA
Asp 141
C.stri_TpdA 152 VVMEGSEELGGEDGLGKLIDANPELFTADVIFIGDGGNVAVGIPTLTTHLRGGAQLRFKV
C.diph_DIP2037 151 FLVEGSEEQGG-AELSDLIKKHPELFDTDVILIADSGNQAVGIPTMTTTLRGGARITVTL
C.jeik_jk0266 164 IVVEGSEERGG-YGLEDLLAEKPELFAADTFLIADSGNDALGEPSLCTALRGSAPVTVRT
P.aeru_Cpg2 168 VLFNPDEETGS---------AGSKQLIAELARQQD--YVFSYEPPDRDAVT-----VATN
E.coli_ArgE 138 ILATADEETS---------MAGARYFAETTALRPD--CAIIGEPTSLQPVR------AHK
E.coli_DapE 127 FLITSDEEASA-------HNGTVKVVEALMARNERLDYCLVGEPSSIEVVG-DVVKNGRR
Glu 176 Glu 200
C.stri_TpdA 212 DTLEGPVHSGGWGGAAPDAAHALIRIIDSFFDEHGRTTIEGVDTTAKWEGDPYDRETFRK
C.diph_DIP2037 210 RTLESGVHSGAFGGAAPDATAALIRLLDTLKDEHGRTTIDGVDCTAHWEGGTYDREAFKK
C.jeik_jk0266 223 RTLAQPMHSGQFGGSAPDALVELVQLLSTLHDENGLVAVPGLEPKERWGGVGPTEQEFRD
P.aeru_Cpg2 212 GIDGLLLEVKGRSSHAGSAPEQGRNAILELSHQLLRLKDLGDPAKG--------------
E.coli_ArgE 181 GHISNAIRIQGQSGHSSDPARG-VNAIELMHDAIGHILQLRDNLKERY-----HYEAFTV
E.coli_DapE 179 GSLTCNLTIHGVQGHVAYPHLADNPVHRAAPFLNELVAIEWDQGNEFFP-----------
C.stri_TpdA 272 DARVLDGVQLLGTVDDEPADMVWARPAITVIGFTSVPVEDATNIVNPTAEAQFNLRVPAP
C.diph_DIP2037 270 DATMLEGTTIMGTENDNPADMVWARPAISIIGFTSTPVDHAINAVPPVASARLNLRVPPK
C.jeik_jk0266 283 NAGVTDGVELYGAGEWQPNDLTVMNPSITITGLDALSVADSVNSVPATAAAVVSLRVPPG
P.aeru_Cpg2 258 --TTLNWTLARGG--------------------------EKRNIIPAEASAEADMRYSDP
E.coli_ArgE 235 PYPTLNLGHIHGG--------------------------DASNRICACCELHMDIRPLPG
E.coli_DapE 228 -ATSMQIANIQAG-------------------------TGSNNVIPGELFVQFNFRFSTE
C.stri_TpdA 332 QSAAEVAKKVEEQIRARAPWGAKVEVSITGVNEPFSTDPNGPAVQHFGKCLQDAYGAE--
C.diph_DIP2037 330 MDANEVANALVEHLKNHVPWGAHIDVTYDDANQPFSAKLDGPAMQLFNSCLAGAYGQD--
C.jeik_jk0266 343 REPQECQDLLVKHLESQKTN-ALVEIERGSLAEAFQADTSGPALQRLGEALGEVYG-K--
P.aeru_Cpg2 290 AESERVLADARKLTGERLVADTEVSLRLDKGRPPLVKN---PASQRLAETAQTLYGRIGK
E.coli_ArgE 269 MTLNELNGLLNDALAP-VSERWPGRLTVDELHPPIPG-YECPPNHQLVEVVEKLLG-A--
E.coli_DapE 262 LTDEMIKAQVLALLEKHQLRYTVDWWLSGQPFLTARGKLVDAVVNAVEHYNEIKP-----
C.stri_TpdA 390 --HLTVVGTGGSIPLTVTLQKHFPDAEFALYGVADPAANIHGVDESVDPTEIEHVAIAEA
C.diph_DIP2037 388 --DTVKIGSGGSIPLCSELLEVVPRAELALFGVEDPQATIHSPNESVDPNEIRDIAVAEA
C.jeik_jk0266 399 --ETMEVASGGSIPLTNKLLGAYPQAELALYGIEEPKCAIHSADESVDPGEIEAIATAEL
P.aeru_Cpg2 347 RIEPIAMRFGTDAGYAYVPGSDKPAVLETLGVVG---AGLHSEAEYLELSSIAPRLYLTV
E.coli_ArgE 324 --KTEVVNYCTEAPFIQTLCPTLVLGPGSIN-------QAHQPDEYLETRFIKPTRELIT
E.coli_DapE 317 --QLLTTGGTSDGRFIARMGAQVVELGPVN-------ATIHKINECVNAADLQLLARMYQ
His 385
C.stri_TpdA 448 EFLLTYGK---
C.diph_DIP2037 446 AFLLSYSK---
C.jeik_jk0266 457 LFLLRTAEAHS
P.aeru_Cpg2 404 ALIRELSAD—
E.coli_ArgE 375 QVIHHFCWH--
E.coli_DapE 368 RIMEQLVA---
Figure S3. Sequence homology of the dipeptidase to other protein sequences. C.stri_TpdA, novel
thiol precursor dipeptidase; C.diph_DIP2037, closest relative in databank, putative metallopeptidase
from Corynebacterium diphteriae; C.jeik_jk0266, thiol precursor dipeptidase homologue in the axilla
bacterium Corynebcaterium jeikeium K411; P.aeru_Cpg2, Carboxypeptidase G2 from Pseudomonas
aeruginosa; E.coli_ArgE, ArgE in E. coli and E.coli_DapE, DapE in E. coli. The Zinc-binding
residues in Carboxypeptidase G2 from P. aeruginosa are underlined and indicated by three letter code
below.
Roger Emter and Andreas Natsch
Corynebacteria
) Conjugate by SSecreted Cys-Gly-(
3-Methyl-3-sulfanylhexan-1-ol from a
the Human Body Odorant
-Lyase Is Required for the Release ofβa
The Sequential Action of a Dipeptidase and
Enzyme Catalysis and Regulation:
doi: 10.1074/jbc.M800730200 originally published online May 30, 2008
2008, 283:20645-20652.J. Biol. Chem.
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