The sequential action of a dipeptidase and a beta-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

Abstract

Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-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 determined. The corresponding gene was cloned and expressed in Escherichia 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 dipeptidase 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.

FIGURE 1.

Proposed scheme for the release of 3M3SH from the Cys-Gly-(S) conjugate.

FIGURE 2.

LC-atmospheric pressure chemical ionization (+)-MS analysis of synthetic Cys- and Cys-Gly-(S) conjugates of 3M3SH (A) and unhydrolyzed axilla secretions fractionated by gel filtration (B). A, a solution containing each 100 μm 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 chromatograms 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.

FIGURE 3.

Release of 3M3SH from synthetic conjugates by bacterial extracts and recombinant β-lyase. Total cell extracts (0.25 mg of protein/ml) of bacterial strains isolated from axillary skin and β-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, Staphylococcus 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 μl of each reaction were spotted on a TLC plate and developed with 1-butanol:acetic acid:H₂O (4:1:1).

FIGURE 5.

GC-FPD analysis of axilla secretions treated with the TpdA and the β-lyase. The aqueous fraction from axilla secretions pooled from two donors was split into four portions, treated with the enzymes (10 μg/ml) for 2 h, extracted with solvent, and analyzed with GC with a sulfur specific detector. A, untreated sample; B, sample treated with the dipeptidase TpdA; C, sample treated with the β-lyase; D, sample treated with TpdA and β-lyase; and E, 2 ppm of synthetic 3M3SH as reference.