From the Givaudan Dübendorf Ltd., Ueberlandstrasse 138, CH-8600 Duebendorf, Switzerland
Received for publication, October 3, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Human axillary odor is known to be formed upon
the action of Corynebacteria sp. on odorless axilla
secretions. The known axilla odor determinant 3-methyl-2-hexenoic acid
was identified in hydrolyzed axilla secretions along with a chemically
related compound, 3-hydroxy-3-methylhexanoic acid. The natural
precursors of both these acids were purified from non-hydrolyzed axilla
secretions. From liquid chromatography/mass spectrometry analysis, it
appeared that the acids are covalently linked to a glutamine residue in
fresh axilla secretions, and the corresponding conjugates were
synthesized for confirmation. Bacterial isolates obtained from the
human axilla and belonging to the Corynebacteria were found
to release the acids from these odorless precursors in
vitro. A Zn2+-dependent aminoacylase
mediating this cleavage was purified from Corynebacterium
striatum Ax20, and the corresponding gene agaA was
cloned and heterologously expressed in Escherichia coli.
The enzyme is highly specific for the glutamine residue but has a low
specificity for the acyl part of the substrate. agaA is
closely related to many genes coding for enzymes involved in the
cleavage of N-terminal acyl and aryl substituents from amino acids.
This is the first report of the structure elucidation of precursors for
human body odorants and the isolation of the bacterial enzyme involved
in their cleavage.
The axilla region of humans contains a dense arrangement of
apocrine, eccrine, and sebaceous glands, and it is an everyday experience, that volatile substances emanating from these areas make a
key contribution to human body odor. Although this odor is perceived by
today's society as mainly unpleasant, several studies indicate that it
may contain chemical signals that affect the menstrual cycle (1) or
that may be involved in a major histocompatibility complex
allele-dependent mate selection (2). These studies point to
an important role of body odors in the evolutionary history of man.
Sweat as it is secreted by axillary glands is odorless. Since the
pioneering work of Shelley et al. (3), it is known that (a) the typical strong axilla odor can only be released from
apocrine secretions, and (b) that the action of skin
bacteria is needed to generate the odoriferous compounds from
non-smelling molecules present in these secretions. Indeed, the axilla
is a skin region supporting a dense bacterial population, which is
dominated by the two genera Staphylococcus and
Corynebacteria (4, 5). Most individuals carry a flora that
is dominated by either one of these two genera, and a strong
correlation was found between a high population of
Corynebacteria and a strong axillary odor formation (4, 6).
As a practical consequence of these findings, halogenated
antibacterials and aluminum preparations for reducing the bacterial
population have become the main active ingredient of commercial
deodorants for the last 40 years. The scientific conclusion from this
early work was that axilla secretions contain non-odoriferous
precursors that must be transformed by bacterial enzymes only present
in Corynebacteria and not in Staphylococci. Considerable progress has been made since then to identify the odoriferous compounds in human body odor, but the biochemistry of axillary odor formation has received relatively little attention. Indeed, no precursor structure has been isolated from axilla secretions and confirmed by re-synthesis, and no specific bacterial enzyme recognizing such a structure has been isolated and reported in the
literature. Notwithstanding, several theories and indirect evidence
have been published.
The focus of early studies was mainly on odoriferous steroids after the
boar pheromone 5 Materials--
Unless otherwise noted, all chemicals were
purchased from Fluka (Buchs, Switzerland). Z-protected amino acids and
peptides as enzyme substrates were from Aldrich and from Senn Chemicals (Dieseldorf, Switzerland).
Collection of Axilla Secretions--
Fresh axilla secretions
were sampled from healthy human donors (27 male and 12 female) by
placing a stainless steel cylinder (internal diameter 3.6 cm) onto the
axilla, adding 4 ml of 10% ethanol and massaging the area with a metal
spatula for 1 min. This procedure does not discriminate between
apocrine and eccrine secretions. The samples were immediately frozen
and concentrated by lyophilization. The samples were redissolved in 400 µl of water and then extracted twice with MTBE and twice with hexane
to remove interfering lipids. At this stage the samples were split, and half was saved for analysis of the potential precursor compounds. For
analysis of the odoriferous components, 50 µl of NaOH (5 M) was added to the remaining half, and the samples were
heated for 20 min at 100 °C. They were then re-acidified by adding
50 µl of HCl (5 M) and applied to a solid phase
extraction C-18 Bond Elute solid phase extraction cartridge (Varian).
The column was washed with H2O and eluted with 50 µl of
MTBE. For analysis, the samples of several donors were pooled.
GC-MS Analysis of Hydrolyzed Axilla Secretions--
For
qualitative GC-MS analysis of the solid phase extraction extracts, a
combination of a Hewlett-Packard 5890 II gas chromatograph and a
Finnigan MAT95 mass spectrometer (low resolution, 70-eV EI mode, ion
source temperature 230 °C) was applied. A DB-5 column (J & W
Scientific) with a length of 60 m, inner diameter of 0.25 mm, and
film thickness of 0.25 µm was used. 1.5 µl of the MTBE solution
were injected in the splitless injection mode (230 °C). The
temperature of the column oven was initially set to 30 °C for 5 min
and subsequently increased to 50 °C (10 °C/min) and then to
250 °C (2 °C/min).
Fractionation and LC-MS Analysis of Aqueous Axilla
Secretions--
The concentrated, pooled, non-hydrolyzed sample was
separated on a Superdex Peptide HR10/30 gel filtration column (Amersham Biosciences) using (NH4)2CO3 (100 mM) as elution buffer. Individual fractions of this
separation step were tested for the content of an axillary odor
precursor by hydrolysis of aliquots in 1 M NaOH. The
fractions developing strong odors upon hydrolysis were subjected to
LC-MS/MS analysis using a Finnigan LCQ mass spectrometer operated in
the atmospheric pressure chemical ionization mode and equipped with a
Flux Rheos 2000 high pressure liquid chromatography pump. High pressure
liquid chromatography separation was performed on a C18 RP
column modified for proteins and peptides (Vydac, Hesperia, CA). The
mobile phase consisted of H2O (A) and MeOH (B) each
containing 1% HOAc (v/v). The solvent flow was 0.25 ml/min and the
following gradient was used: 0-1 min, 100% A; 1-6 min from 100% A
to 100% B, 6-11 min 100% B, 11-13 min from 100% B to 100% A. MS/MS spectra were recorded with 30% relative collision energy.
Synthesis of Reference Compounds--
The precursor structures
3M2H-Gln and HMHA-Gln were prepared by the coupling of the activated
sweat acids (N-hydroxysuccinimide ester) with
L-glutamine. Whereas 3M2H is commercially available (Narchem Corp., Chicago), 3-methyl-3-hydroxyhexanoic acid (HMHA) was
synthesized in a Zn2+-mediated reaction of ethyl
bromoacetate with 2-pentanone, followed by saponification in ethanolic
sodium hydroxide solution. Zn2+ powder in cyclohexane was
heated to reflux and activated with a little ethyl bromoacetate. Then a
mixture of ethyl bromoacetate and 2-pentanone was added dropwise. After
2 h of reflux the mixture was allowed to stand overnight and
worked up in the usual manner. The resulting ethyl ester was
dissolved in ethanol, treated with aqueous sodium hydroxide (2 N), and heated to reflux for several hours to yield the
desired 3-methyl-3-hydroxyhexanoic acid, after acidification with
aqueous hydrochloric acid and extraction with ether.
The acids and N-hydroxysuccinimide were dissolved in
dioxane, and a solution of 1,3-dicyclohexylcarbodiimide in dioxane was added dropwise under ice-water cooling. The resulting suspension was
allowed to warm to ambient temperature and stirred for 24 h.
1,3-Dicyclohexylurea was filtered, and the filtrate was concentrated to
give the corresponding N-hydroxysuccinimide ester. This was dissolved in dioxane and added dropwise to a solution of
L-glutamine and triethylamine in water. After stirring for
24 h, work up in the usual way furnished the compounds 3M2H-Gln
and HMHA-Gln.
Isolation of Bacterial Strains and Culture Conditions--
For
isolation of axilla bacteria, the same method as described above for
sampling axilla secretion was applied, with the exception that
phosphate buffer, pH 7, containing 1% Tween 80 was used as the
sampling liquid. The samples of axilla washings were spread plated on
tryptic soy agar (Difco) amended with 5 g/liter Tween 80 and 1 g/liter
lecithin. Single isolates obtained after 48 h of incubation were
subcultured and characterized. Strains that had a coccoid cell
morphology were identified with the ID Staph 32 kit (BioMerieux,
France) and strains with a typical coryneform morphology (short,
irregular rods) with the API coryne kit (BioMerieux, France). Gram
reaction was determined with the KOH string technique (17), and
lipophilicity was determined based on differential growth on media with
or without Tween 80 as lipid source. For enzymatic assays and enzyme
purification, the strains were grown overnight in Mueller-Hinton broth
(Difco) amended with 0.05% Tween 80. As a reference strain
Corynebacterium xerosis (DSMZ 20170; obtained from the
German type strain collection) was used.
Enzyme Assays--
To evaluate enzymatic activity in intact
bacteria, an overnight culture was harvested by centrifugation and
resuspended to a final A600 of 1.0 in a
semisynthetic medium (per liter: 3 g of
KH2PO4, 1.9 g of
K2HPO4, 0.2 g of yeast extract, 0.2 g
of MgSO4 × 7 H2O, 1.4 g of NaCl, 1 g
of NH4Cl, 10 mg of MnCl2, 1 mg of FeCl3, 1 mg of CaCl2). Aliquots of this
stationary culture were then amended with a final concentration of 2 mM HMHA-Gln, 3M2H-Gln, or alternative substrates (160 mM stock solution dissolved in Me2SO).
After 24 h of incubation (with shaking at 300 rpm; 36 °C), the
samples were acidified with HCl and extracted with an equal volume of
MTBE, and the amount of released acids was determined by GC as
described above. No HCl was added if carbamates (e.g. Z-Gln)
were used as substrates. To evaluate activity in cellular extracts,
cells were washed, resuspended in as small volume of Buffer A (50 mM NaCl; 50 mM
NaH2PO4/K2HPO4; pH 7),
amended with a 10-fold volume of glass beads (425-600 µm, Sigma),
and mechanically disrupted by vortexing them at maximal speed for 30 min. The lysates were centrifuged and the supernatants saved and
diluted, and enzymatic assays were run with these crude extracts.
Alternatively, enzyme assays were also done using the pure enzyme in
Buffer A, either purified from the wild-type strain
Corynebacterium Ax20 or from the recombinant strain (see
below). As an alternative to the GC detection of the released acid, a
fluorescent test was used. The enzyme reaction was stopped by adding
0.5 volumes of acetonitrile containing 2.5 mM fluorescamine
(Fluka, Switzerland) thus derivatizing the free amino group of the
released L-glutamine. Fluorescence was then measured with
an LS-50 fluorescence spectrometer (PerkinElmer Life Sciences) with an
excitation wavelength of 381 nm and an emission wavelength of 470 nm.
Purification of the Aminoacylase--
Corynebacterium
striatum Ax20 was selected to isolate and purify the enzyme
responsible for the cleavage of HMHA-Gln. Cell lysates from 2-liter
cultures were prepared as described above, and the proteins were
fractionated by precipitation with an increasing concentration of
(NH4)2SO4. The precipitate obtained
between 50 and 80% saturation of
(NH4)2SO4 contained the active
enzyme. This enriched fraction was dissolved in Buffer A and desalted
by repetitive dilution and concentration by ultrafiltration (Amicon
membrane YM10, Millipore, Bedford, MA). The sample was then
sequentially passed over four chromatography columns as follows: 1)
DEAE-Sepharose CL-6B anion exchange resin (Amersham Biosciences),
elution with a linear gradient from 0 to 800 mM KCl in
Buffer A; 2) phenyl-Sepharose hydrophobic interaction resin (Amersham
Biosciences), elution with a linear gradient from 1000 to 0 mM (NH4)2SO4 in Buffer
A; 3) Mono Q strong anion exchange column on the fast protein liquid chromatography system (Amersham Biosciences), elution with a gradient from 0 to 800 mM KCl in Buffer A; and finally Mono P weak
anion exchange column on the fast protein liquid chromatography,
elution with a gradient from 0 to 800 mM KCl in a 50 mM bis-Tris buffer instead of Buffer A. After each column
separation the active fractions (determined by the fluorescent assay)
were pooled and desalted by dilution/ultrafiltration. The resulting
apparently pure enzyme was subjected to tryptic digestion, and the
sequence of two internal peptides was determined with LC-ESI-MS/MS
analysis, and the N terminus was determined by automated Edman
degradation. The molecular weight of the isolated enzyme was determined
with nano-ESI-MS.
Protein Determination and SDS-PAGE--
Protein concentrations
were determined with the Bradford reagent (Bio-Rad) using bovine serum
albumin as standard. SDS-PAGE was performed according to Laemmli (18)
with 4% stacking gels and 9% separation gels. Protein bands were
detected by Coomassie Blue staining.
Molecular Biology Methods--
Chromosomal DNA of Ax20 was
obtained from cell lysates by proteinase K digestion and subsequent
extraction with hexadecyl-trimethylammonium bromide/NaCl and
chloroform/isoamyl acetate (19). Based on the partial amino acid
sequence analysis of the aminoacylase, degenerated primers were
designed to amplify fragments between the N terminus and the internal
peptide sequences. Standard PCR conditions were used according to the
manufacturer (Taq polymerase, Sigma). The annealing
temperatures were optimized on a gradient cycler (T-Gradient, Biometra,
Göttingen, Germany). The amplified DNA was cloned into the vector
pGEM-T Easy (Promega, Madison, WI), and the nucleotide sequence was
determined on the ABI-Prism model 310 (PE Biosystems, Rotkreuz,
Switzerland) using standard methods. Based on the obtained partial
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 Laboratories, Palo
Alto, CA). The upstream and downstream regions were then amplified as
described in the instructions to the Universal GenomeWalkerTM kit
(Clontech Laboratories, Palo Alto, CA) and cloned
into the vector pGEM T-easy, and the nucleotide sequence was
determined. The full-length sequence of the open reading frame coding
for the enzyme was then amplified with PCR from chromosomal DNA of Ax20
using the specific primers 5'-CAT GCC ATG GCA CAG GAA AAT TTG CAA and
5'-CCC AAG CTT TCA CTT CAT CAA CCA GGG CG. The amplified DNA fragment
was digested with NcoI and HindIII and ligated
into the vector pBAD/gIIIA (Invitrogen) pre-digested with the same
enzymes. The resulting plasmid pBAD/gIIIA-AMRE was transformed into the
host strain E. coli TOP10 (Invitrogen). This strain was
grown in LB broth until it reached an A600 of 0.5. The culture was induced with arabinose (0.2% final
concentration), incubated for 4 h, harvested by centrifugation,
and disrupted by ultrasonication. The extracts were used to purify the
recombinant enzyme as described above but only with two chromatographic
steps, namely phenyl-Sepharose and Mono Q. Data base comparisons of the deduced open reading frame were made to public protein sequence data
bases (Swiss-Prot and GenBankTM bacterial sequences and to
data bases containing the genomes of different bacteria) using the
gapped blast algorithm (20) on the Biology WorkBench (biowb.sdsc.edu) platform.
Analysis of Hydrolyzed Axilla Secretions--
The organic extracts
obtained from axilla secretions before hydrolysis were almost odorless,
and only upon hydrolysis and re-acidification was a strong axillary
odor developed, indicating that the volatiles were mainly present in
the collected secretions as covalently linked, water-soluble
precursors. The organic phase obtained from extraction of the
hydrolyzed and re-acidified samples of axilla secretions was therefore
first olfactorily evaluated with a gas chromatograph equipped with a
sniff port, and peaks of effluents which exhibited typical
axillary odor were marked on the chromatograms. These peaks were then
further analyzed by GC-MS. A typical chromatogram is shown in Fig.
1a. The presence of 3M2H could
be confirmed to be an important odoriferous component (retention
time 30.8 min) based on mass spectrum and retention time compared with
a synthetic sample. At a retention time of 37.4 min a second and larger
peak with a hypothetical molecular weight of 146 was found. The mass
spectra of this unknown peak and that of 3M2H are shown in Fig. 1,
b and c. Based on the fractionation pattern and
comparisons to MS data bases, this new compound was proposed to be a
hydrated derivative of 3M2H, namely 3-hydroxy-3-methylhexanoic acid
(HMHA). This latter compound was synthesized, and its retention time
and mass spectrum were found to be identical with the one of the
unknown peak (data not shown). In addition, the hydrolyzed axilla
secretion samples contained small amounts of (Z)-3 methyl-2 hexenoic acid and (E)-3-methyl-3-hexenoic acid based on MS
analytics of the corresponding peaks (data not shown).
Analysis of Non-hydrolyzed Axilla Secretions--
Non-hydrolyzed
samples of axilla secretions were subjected to gel filtration on a
Superdex Peptide HR10/30 column. The resulting chromatogram of the
pooled samples of eight donors is shown in Fig.
2. The fraction at an elution volume of
17.7 ml (of 24 ml total gel bed size) was found to release a strong
odor upon hydrolysis. This elution volume is between the peaks for
Gly6 and Gly3 according to the
manufacturer. The corresponding peak does not absorb at 280 nm. The
typical axilla secretion proteins did elute early (7 ml of elution
volume) as determined by SDS-PAGE (data not shown), and this fraction
released no perceivable odor upon hydrolysis. Thus, these data indicate
that the precursor is not a protein but a smaller molecule in the range
of 200-400 Da. The presence of 3M2H and HMHA was verified in a
hydrolyzed aliquot of the fraction of interest by GC-MS analysis (data
not shown). This fraction was then subjected to LC-MS analysis. It
contained one peak with a pseudo-molecular ion at m/z 257 and a peak with a pseudo-molecular ion at m/z 275. Assuming
an ester or amide linkage of the two acids, the molecular masses of 256 and 274 suggested that 3M2H and HMHA are bound to a molecule with
molecular mass of 146. Several common biological molecules with this
mass were considered, and glutamine was found to fit best the MS
fractionation pattern observed. This led to the hypothesis that 3M2H is
linked to the N Isolation of Axilla Bacteria Able to Cleave HMHA-Gln and
3M2H-Gln--
The axilla flora of 21 donors was isolated on tryptic
soy agar, and single isolates obtained after 48 h of incubation
were subcultured and identified. A total of 19 individual strains of Corynebacterium and 25 strains of Staphylococcus
were obtained. Stationary cultures of the strains were amended with
HMHA-Gln or 3M2H-Gln and analyzed after a 24-h incubation for released acids. Isolates from both the non-lipophilic C. striatum and
the lipophilic Corynebacterium jeikeium and
Corynebacterium bovis, but not all
Corynebacterium, released a significant quantity of 3M2H and
HMHA, whereas none of the strains of Staphylococcus sp. or
Micrococcus sp. exhibited this enzymatic activity. Table
I gives the results for a subset of the
strains tested. These data are in good agreement with the well known
fact that only subjects with an axilla flora dominated by
Corynebacteria produce the most typical axillary odor. Thus,
N Characterization of the N Purification of the Aminoacylase and Cloning of the Corresponding
Gene--
The glutamine-specific aminoacylase was purified from the
cellular extracts of C. striatum Ax20 according to the
scheme described under "Experimental Procedures." The aminoacylase
activity eluted from all the columns as a single peak indicating that
only one enzyme is involved in cleavage of these substrates. The
isolated enzyme was stable at 4 °C, and it was over 90% pure when
analyzed by SDS-PAGE with one major band with an apparent
molecular mass of 48 kDa. Its effective molecular mass was
determined by nano-ESI-MS analysis and found to be 43,365 ± 5 Da.
The N-terminal sequence was found to be
AQENLQKIVDSLESSRAEREELYKWFHQHPEMSMQE, and LC-ESI-MS/MS analysis after
tryptic digestion revealed the sequences DLWSGTFIAVHQPGEEIGGTK and
WGWSGFAAGSDAPGN of two internal peptides. Based on each of these
peptides, four degenerated oligonucleotides were designed, and a total
of 32 primer combinations between the internal peptides and the
N-terminal sequence were used for PCR amplification with chromosomal
DNA of Ax20 as template. Six primer combinations gave a major band of
350 and 650 bp, respectively, whereas the others gave no or unspecific
amplification. These amplicons were cloned, and the sequence was
determined. Specific nested oligonucleotides were then designed to
clone the upstream and downstream region using the Universal
GenomeWalkerTM kit (Clontech
Laboratories, Palo Alto, CA) with libraries generated from
PvuII and SmaI digests of chromosomal DNA. An
upstream fragment of 1200 bp and a downstream fragments with 3000 bp
were obtained. Within this sequenced region (4600 bp), an open reading
frame coding for a polypeptide with 399 amino acid residues and
with a high homology to known aminoacylases, some carboxypeptidases, and various putative peptidases was identified. The calculated molecular mass of the deduced polypeptide starting from the
experimentally determined N terminus is 43,365 Da, corresponding well
to the value measured with nano-ESI-MS analysis. The gene is named
agaA (for acylglutamine-aminoacylase) and the complete
sequence was deposited in GenBankTM.
Sequence Comparison to Related Proteins--
The protein deduced
from the open reading frame has a very high homology to a large number
of bacterial sequences, although the function of most of these genes
has not been determined experimentally. Indeed, when comparing the
sequence to 15 different bacterial genomes (Streptococcus
pneumoniae R6, Staphylococcus aureus Mu50, Pseudomonas aeruginosa,
Neisseria meningitidis, Mycobacterium tuberculosis, Listeria
monocytogenes, Helicobacter pylori, Haemophilus influenzae Rd, E. coli
K-12, Corynebacterium glutamicum, Clostridium perfringens,
Campylobacter jejuni, Borrelia burgdorferi, Bacillus subtilis, and
Agrobacterium tumefaciens), 11 of them, both Gram positives
and Gram negatives, contain at least one gene with a significant
homology (E value <1 Characterization of the Pure Recombinant Enzyme--
A DNA
fragment containing the complete open reading frame was then cloned
into the expression vector pBAD/gIIIA and transformed in E. coli TOP10. The transformants produced a high yield of the protein, and it was purified to >95% purity with two chromatographic steps (Fig. 6). The recombinant enzyme
was similarly inhibited by Zn2+-chelating agents, and it
exhibited the equal substrate specificity toward acylated amino acids
as initially observed in the crude cell extracts (data not shown). A
subset of the substrates was then used to determine enzyme kinetics.
The kinetic parameters are listed in Table
III, and the Michaelis-Menten plot is
shown in Fig. 7. Among the substrates
tested, N Several short, 3-methyl-branched fatty acids were found to be
released from odorless sweat upon hydrolysis. This confirms the finding
of Zeng et al. (13) showing the important contribution of
this class of molecules to the olfactive impression of axilla secretions. The nature of the precursor structures found in this work
is in very good agreement with earlier observations (16) suggesting
that a bond sensitive to alkaline hydrolysis must be involved and that
the putative precursor is water-soluble. In addition, the isolation of
an odorless precursor confirms the observation made 50 years ago that
fresh axilla secretions are odorless (3). The fact that bacteria, but
only Corynebacteria, cleave the precursor further confirms
the large number of reports (e.g. Refs. 4 and 6) showing
that a dense population of coryneforms is a prerequisite for strong
odor formation, and this is the first report clearly proving a
biochemical mechanism explaining these observations. However, the
nature of the precursor identified in this work at first sight is in
contradiction to the work of Zeng et al. (15) and Spielman
et al. (28) showing association of 3M2H with apolipoprotein
D in axilla secretions, and suggesting that this protein-fatty acid
association indeed is the precursor. Nevertheless, once cleaved from
the glutamine residue as shown in this work, the acids might
subsequently associate non-covalently with apolipoprotein D, and this
non-covalent association could be just one more mechanism of slow
release of these volatiles from the axilla. A similar release mechanism
is postulated for hamster aphrodisin and a putative hydrophobic
pheromonal compound (29). Finally, apoD has an N-terminal glutamine
residue (15), and we cannot rule out the possibility, that a covalent
linkage to this residue exists and that the proteins are first cleaved by an endopeptidase thus releasing the terminal acid-Gln conjugate. In
this case, the accumulation of a significant amount of 3M2H-Gln or
HMHA-Gln in axilla secretions as shown in this work and the lack of
odor released from the protein fraction by hydrolysis would indicate
that cleavage of the glutamine conjugates is the rate-limiting step.
However, such a mechanism would require that for the release of each
volatile molecule of Mr 128 or 146, a complete
protein molecule with Mr 23,000 needs to be
synthesized and secreted.
It has been speculated that body odor formation originates from common
catabolic pathways of the skin bacteria and therefore is just a
by-product of bacterial metabolism of unspecific skin secretions, which
themselves are by-products of the metabolism of the body. Examples of
such catabolic processes put forward are the formation of isovaleric
acid from L-leucine or the formation of short acids by
incomplete degradation of skin lipids (30). The nature of the
identified 3M2H-Gln and HMHA-Gln points in another direction. Indeed it
is rather unlikely that these compounds are by-products of the human
metabolism, and it appears more likely that they are synthesized
specifically to exert their action once secreted in the axilla region.
The actual benefit of the secretion in a precursor form instead of
direct secretion of the acids could be manifold. On the one hand, a
precursor leads to a controlled release making the chemical signal more
long lasting. On the other hand specific secretion into the ducts of
the apocrine glands could pose physiological problems for the low
molecular weight acids that are overcome by a water-soluble precursor structure.
The fact that enzymes of human skin bacteria specifically recognize the
precursor structures indicates that the bacteria have adapted their
enzymes to the specific axilla secretions and points to a very close
interaction and co-evolution of humans and their axilla flora. The
aminoacylase described in this work is unique in its substrate
specificity, being very selective for the glutamine residue but having
a very broad substrate specificity regarding the acyl part. This
specificity would open up the possibility that other potential
Gln-containing precursor structures in sweat could be cleaved by the
same enzyme. Other branched acids have been reported in axilla
secretions (13), and we could confirm this finding (data not shown).
Whether some of these acids are also linked to Gln in axilla secretions
remains to be investigated. Indeed, the Km values
for the substrates N Closely related enzymes are present in the majority of the bacterial
species (Gram-negatives, Gram-positives, and Archaea) and in plants.
Most of them have not been investigated for their substrate
specificity, but in all instances where the substrate specificity is
known, these enzymes catalyze the release of an aliphatic or aromatic
acid from the amino group of an amino acid and/or they cleave
carbamates (21, 23-27). Besides this common denominator, the substrate
specificity of these enzymes varies widely, and the different enzymes
depend on different metal co-factors (manganese, cobalt, or zinc). The
very high specificity for one particular amino acid residue has not
been found for most of the related enzymes, which cleave a broad range
of synthetic substrates. However, the physiological substrate is not
known for most of the enzymes, especially those isolated form bacteria.
The conserved sequences in this family of enzymes do not contain either
of the common denominators HEXXH, HXXEH,
HXXE, or HXH found in many
zinc-dependent metallopeptidases (31), and thus the active
site configuration and the residues involved in Zn2+
chelation remain to be determined. The strikingly high conservation of
the two motifs RADXDALP and MHACGHDXH among
species belonging to the Archaea, Eubacteria, and
plants is especially interesting in this respect and deserves further attention.
Finally, the elucidation of a specific biochemical mechanism of human
body odor formation brings back the question about its biological
function. Although the role of chemical communication in animals has
been studied in detail, the information on humans is scarce. Today,
body odors are mainly perceived as offensive and negative. This might
be the result of social learning but could also reflect the actual
function that these compounds had in our evolutionary past. They may
have always been involved in marking dominance and repelling rivals,
although both the popular and the scientific discussion on the
potential semi-chemical activity of human body odor has mainly focused
on its potential attractiveness and importance in mate selection (2).
In humans, a functional vomeronasal organ for perceiving pheromones
cannot be found in adults. Whereas the family of genes coding for
vomeronasal receptors contains up to 300 genes in rodents, only one is
expressed in humans, and the remainder are lost or are pseudogenes
(32). Thus it is likely that chemical communication in man solely
depends on olfaction, and a more detailed understanding of chemical
composition, biochemical formation, and function of body odor still
deserves further attention.
From a practical point of view, the findings of this work also point to
a novel way to combat body odors. Today prevention is based on the
daily topical application of (mainly halogenated) antibacterial agents
and aluminum salts. These agents unspecifically and transiently reduce
the bacterial skin flora. The fact that an enzyme related to the class
of metallopeptidases with a unique substrate specificity plays an
important role in body odor formation points to the possibility of
synthesizing specific inhibitors, because for metallopeptidases a
rational inhibitor design has been successfully applied in many
instances (33). Such compounds could be used as additional or
alternative active ingredients in deodorants.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-androst-16-en-3-one (7, 8) and a related
odoriferous steroid 5
-androst-16-en-3
-ol (9) had been detected in
human axilla secretions. Nevertheless, androstenone is only perceived
by 50% of the human population (10) and thus cannot be the main
odoriferous component. Release of androstenol from secreted
sulfates and glucuronides in the axilla was proposed to be mediated by
bacterial enzymes (11). Nevertheless, the steroid precursors were not
isolated, and clear evidence for corynebacterial enzymes cleaving
sulfate and glucuronide conjugates of steroids was not presented,
although arylsulfatase and aryl glucuronidase activity was found in
these bacteria. The involvement of thiols as key odor components and
the putative implication of a pyridoxal phosphate-dependent
-lyase (probably related to cystathionine
-lyase) in the release
of these thiols has been proposed (12), but without data on the
structure of the secreted precursor or isolation of the bacterial
enzyme. A very thorough analysis of the chemical composition of
axillary odor was presented by Zeng et al. (13). They
proposed that short, branched fatty acids make a major contribution
with (E)-3-methyl-2-hexenoic acid
(3M2H)1 being the key odor
component. This compound had initially been found in sweat of
schizophrenic patients (14), and whether its presence is a specific
marker of schizophrenia was the subject of a longer debate. Zeng
et al. (13) could clearly show its contribution to axillary
odor of healthy donors. They could also show (15) that it is
non-covalently associated with apolipoprotein D, the major protein
present in axilla secretions. Furthermore, they showed that 3M2H is
released upon incubation of the aqueous fraction of apocrine secretions
with Corynebacteria or hydrolysis with NaOH, indicating a
water-soluble, covalent precursor must be present (16). Yet its
structure and the bacterial enzyme involved in its cleavage were not
described. Here we report the isolation of a new branched fatty acid
related to 3M2H and covalent linkage of both acids to
L-glutamine in human axilla secretions. Furthermore, a
specific zinc-dependent aminoacylase, which triggers the
release of these odoriferous components from odorless axilla secretions, was purified from axilla isolates of
Corynebacteria, characterized, and heterologously expressed
in Escherichia coli.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GC-MS analysis of hydrolyzed axilla
secretions. A pooled sample from nine individual donors is shown.
a, the total ion count; b, the mass spectrum
recorded at a retention time of 30.8 min corresponding to 3M2H;
c, the mass spectrum recorded at a retention time of 37.4 min corresponding to HMHA.
atom of L-glutamine
(i.e.
N
-3-methyl-2-hexenoyl-L-glutamine-;
3M2H-Gln), whereas the second peak with a mass of 274, based on its
mass and fractionation pattern, corresponds to the hydrated analogue
N
-3-hydroxy-3-methylhexanoyl-L-glutamine
(HMHA-Gln). These two compounds were then synthesized, and their MS
spectra and retention times in the LC-MS/MS analysis were compared and
found to be identical to the compounds present in axilla secretions.
The presence of these compounds in the hydrophilic phase of
non-fractionated axilla secretions of individual donors was then also
verified. In Fig. 3b, the
LC-MS/MS analysis of such a sample is shown, and the analytical data
for the two synthetic references are given in Fig. 3a. The synthesized compounds partly released the acids upon hydrolysis with
NaOH and re-acidification which perfectly fits the observations made
previously by us and others (16) on the nature of the precursor. Thus,
from all this analytical evidence one could predict the enzymatic
activity needed for the release of the odoriferous component, namely a bacterial N
-acylglutamine
aminoacylase, and the corresponding scheme and structures are shown in
Fig. 4.
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Fig. 2.
Gel filtration chromatogram of non-hydrolyzed
aqueous fraction of axilla secretions. Amersham Biosciences
Peptide HR 10/30 column was used with 24-ml total bed volume,
(NH4)2CO3 (100 mM)
elution buffer. The absorbance of the eluate at 214 nm is shown in
black, and the absorbance at 280 nm is shown in
gray.
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Fig. 3.
LC-MS/MS analysis of axilla secretions.
a, a mixture of the synthetic HMHA-Gln and 3M2H-Gln (each 50 ppm); b, the aqueous phase of an axilla secretion sample.
a-1 and b-1, the total ion chromatogram;
a-2 and b-2, the extracted mass chromatogram at
m/z = 275; a-3 and
b-3, the extracted mass chromatogram at
m/z = 257; a-4 and
b-4, the MS2 spectrum of the peak at 7.3-7.5
min retention time corresponding to HMHA-Gln; and a-5 and
b-5, the MS2 spectrum of the peak at 8.3-8.5
min retention time corresponding to 3M2H-Gln.
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Fig. 4.
Proposed scheme for the release of
odoriferous acids by skin bacteria.
-acylglutamine aminoacylase activity is
indeed present in bacteria isolated from the human axilla, as predicted
based on the identification of the precursor structure. If
N
-lauroyl-L-glutamine was used as
substrate in the same experiment, it was found that also other
Corynebacteria and some Staphylococci can release
lauric acid from this substrate, indicating that even more skin
bacteria may have a related aminoacylase activity more specific for
substrates substituted with a straight chain acid, and thus not
directly involved in the formation of the typical odor of the branched
chain acids.
Cleavage of N-acylglutamine derivatives by axilla bacteria
-Acylglutamine Aminoacylase
Activity--
C. striatum Ax20 was selected to characterize
the aminoacylase activity in more detail. No activity was observed in
the supernatants of stationary phase cultures. On the other hand, the
enzymatic activity could be obtained in the soluble fraction after
mechanical disruption of the cells and subsequent removal of cell
debris by centrifugation. This indicates that the cleavage is mediated by an intracellular, soluble enzyme that is not, or only loosely, associated with the cell membrane. Further experiments were thus done
with cell extracts. The enzymatic activity was lost if these extracts
were preincubated with 0.5 mM of EDTA or
o-phenanthroline, but no inhibition was observed if an
excess of Zn2+ was simultaneously added. Only partial loss
of activity was observed with the combination Mn2+ and
chelating agents. Activity was also lost in presence of 0.5 mM dithiothreitol. The extracts retained their activity if
treated with the serine protease inhibitors phenylmethylsulfonyl
fluoride and Pefablock (4-(2-aminoethyl)-benzenesulfonyl fluoride)
or with pepstatin. Taken together, these observations indicate that the aminoacylase is related to the Zn2+-dependent
metallopeptidases. In the next step, an analysis of the substrate
specificity in the cellular extracts was made. As mentioned above, in
addition to the physiological substrates, N
-lauroyl-Gln was efficiently cleaved also.
Furthermore, carbamates of glutamine were alternative substrates, in
particular carbobenzyloxy-L-glutamine (Z-Gln) as well as
some dipeptides with C-terminal Gln. Among other Z-substituted or
lauroyl-substituted natural amino acids, only a very weak activity for
Z-Ala was observed. No activity for Z-L-asparagine,
Z-L-aspartate,
N-lauroyl-L-aspartate,
Z-L-glutamate, and other substituted essential amino acids
was present in the cell extracts nor was there any cleavage of
Z-D-glutamine, the methyl ester of
Z-L-glutamine or Z-Gln-Gly (Table
II). Thus the enzymatic activity is very
specific for the glutamine residue; it is stereospecific and needs the
free carboxy group of Gln as is typical for carboxypeptidases, and it
has a low specificity for the acyl part of the substrates. Based on the
substrate specificity, it belongs to the class of enzymes summarized in
the official nomenclature under the number EC 3.5.1.14.
Substrate specificity of the enzymatic activity in cellular
extracts of C. striatum Ax20
20 in the gapped blast
comparison). All these genes have the highly conserved sequence
MHACGHDXH and a second conserved motif with a consensus of
RADXDXLPXXE. In addition, they contain two more highly conserved histidines, four glutamates, and one aspartate. The
open reading frame was also compared with genes with a known function
only, and the alignment with the closest relatives with known function
is shown in Fig. 5. The same conserved
motifs as summarized above are also found in this alignment. The gene
cpsA from Sulfolobus solfataricus codes
for a Zn2+-dependent carboxypeptidase able to
cleave benzyloxycarbonylated amino acids similar to the aminoacylase
described in this work but without a high specificity for the amino
acid residue (21, 22). The gene amaA codes for an
aminoacylase in Bacillus stearothermophilus able to cleave
N-acyl derivatives especially of aromatic amino acids (23).
hipO codes for hippuricase in C. jejuni (24) an enzyme present in many bacterial species and hydrolyzing
hippurate (benzoyl-glycine). The related hyperthermostable
carboxypeptidase/aminoacylase from Pyrococccus horikoshii
OT3 has both carboxypeptidase activity especially for terminal Phe and
aminoacylase activity for acetylated amino acids without strict
specificity (25). Finally, ILL2 is one of six ILR1-like genes from
Arabidopsis thaliana (26, 27). These genes code for enzymes
cleaving the phytohormone indole-3-acetic acid from amino acid
precursors. All the six related plant genes share all the conserved
motifs with the bacterial sequences as described above. Based on the
sequence comparison, the aminoacylase belongs to the peptidase family
m40, also known as the ama/hipo/hyuc family of hydrolases.
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Fig. 5.
Sequence homology of the new aminoacylase to
enzymes with known function. Matching sequences were obtained from
GenBankTM bacterial sequences and Swiss-Prot using the
Biology Workbench platform and the BLAST function. Sequences were
aligned to each other using the ClustalW algorithm. N-AGA,
N-acyl-Gln aminoacylase, this work; ph_cp,
carboxypeptidase from Pyrococcus horikoshii (25),
GenBankTM accession number AB009503; CBP1_SULSO,
carboxypeptidase from S. solfataricus (22), EMBL accession
number Z48497; ILR1_ARATH, indole-3-acetic acid-conjugate
hydrolase from A. thaliana (26), Swiss-Prot accession number
P54968; HIPO_CAMJE, hippuricase from C. jejuni
(24), Swiss-Prot accession number P45493; and AMAA_BACST,
aminoacylase from B. stearothermophilus (23),
GenBankTM accession number X74289.
-decanoyl-Gln has the highest
Vmax, and it has a high affinity to the enzyme
(Km of 0.08 mM). Interestingly, the main natural substrate isolated from axilla secretions, HMHA-Gln, has a much
lower affinity (Km = 0.74 mM) but also a
high Vmax, whereas the natural substrate
3M2H-Gln as well as the synthetic carbamate Z-Gln have a significantly
lower Vmax.
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Fig. 6.
Purification of the recombinant aminoacylase
from cell extracts of E. coli TOP10 transformed with
(pBAD/gIIIA-AMRE). 1st lane, 0.5 µl of active fraction
from the Mono Q separation step; 2nd lane, molecular size
marker; 3rd lane, 20 µl of the same active fraction.
Kinetic data for the pure recombinant glutamine-aminoacylase
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Fig. 7.
Kinetic analysis of the recombinant
aminoacylase. The following substrates were tested at varying
concentrations: HMHA-Gln (open squares); lauroyl-Gln
(filled squares); decanoyl-Gln (filled
triangles); 3M2H-Gln (crosses), and Z-Gln (open
triangles).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-decanoyl-Gln and
N
-lauroyl-Gln not found in axilla secretions
are clearly lower then for the natural substrates detected in the
axilla secretions. The high Km values for the
natural substrates could point to a suboptimal cleavage of HMHA-Gln and
3M2H-Gln in the axilla, because the maximal level detected on the skin
of HMHA-Gln was 0.95 nmol/cm2 (data not shown). Depending
on the water film present on the skin, the actual concentration in
sweat is then estimated to be 0.019-0.095 mM and thus
clearly below the Km. However, at a concentration of
0.031 mM HMHA-Gln, a diluted stationary phase culture of
C. striatum (A600 = 0.25) cleaves
10.5% of the substrate within 15 min (data not shown), which indicates
that despite the high Km value, the enzyme may
release significant amounts of HMHA under the physiological conditions
in the axilla.
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ACKNOWLEDGEMENTS |
---|
We thank S. Derrer for synthesizing reference compounds, B. Schilling for critical discussions, A. Heimgartner and M. Wasescha for laboratory assistance, E. Senn for GC-Snif analysis, and H. Lahm (Hoffmann-La Roche, Basel, Switzerland) for amino acid sequence analysis.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" 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 GenBankTM/EBI Data Bank with accession number(s) AF534871.
To whom correspondence should be addressed. Tel.: 41-1-824-21-05;
Fax: 41-1-824-29-26; E-mail: andreas.natsch@givaudan.com.
§ Present address: Hoffmann-La Roche, Basel, Switzerland.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210142200
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ABBREVIATIONS |
---|
The abbreviations used are:
3M2H, (E)-3-methyl-2-hexenoic acid;
MTBE, methyl-tert-butyl-ether;
HMHA, 3-methyl-3-hydroxyhexanoic
acid;
3M2H-Gln, N-(E)-3-methyl-2-hexenoyl-glutamine;
HMHA-Gln, N
-3-methyl-3-hydroxy-hexanoyl-glutamine;
bis-Tris, bis-(2-hydroxyethyl)-amino-tris-(hydroxymethyl)-methane;
LC-MS, liquid chromatography/mass spectrometry;
GC-MS, gas
chromatography-mass spectrometry;
ESI-MS, electrospray ionization-mass
spectrometry;
Z, carbobenzyloxy.
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REFERENCES |
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