The Molecular Basis of Thioalcohol Production in Human Body Odor

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Discovery of a unique C-S lyase involved in the formation of body odour

By screening a range of axillary Staphylococcus species and strains, we identified those coagulase negative staphylococci (CoNS) able to take up and convert Cys-Gly-3M3SH to 3M3SH (Fig. 1B and Supplementary Information Figure S2A). Among these, a monophyletic group of CoNS emerged as the most efficient biotransfomers of Cys-Gly-3M3SH (Fig. 1B Group 1 [G1]), along with Staphylococcus species from two other distinct clades (Fig. 1B Groups 2 and 3 [G2&G3]). Strikingly, Staphylococcus epidermidis, the dominant staphylococcal species present on the skin including the axilla14, does not metabolise Cys-Gly-3M3SH (Fig. 1B, Supplementary Information Figure S2B and C), and nor do other species of human associated staphylococci such as Staphylococcus capitis and Staphylococcus aureus (Fig. 1B; Supplementary Information Figure S2C). The G1 clade contains S. hominis, a species which is strongly associated with body odour, along with Staphylococcus lugdunensis and Staphylococcus haemolyticus, which have been previously linked to thioalcohol production3. In order to elucidate the molecular basis for this highly limited phenotype in staphylococci, we searched staphylococcal genomes for enzymes likely to be involved in the generation of volatile thioalcohols. All staphylococcal genomes encode a DtpT orthologue, involved in precursor uptake15 and PepA, the likely peptidase required for removal of the glycine from the Cys-Gly-3M3SH (Fig. 1A, B), so we reasoned that the lyase step would be unique. Cleavage of Cys-3M3SH to produce 3M3SH involves a β-elimination from an amino acid substrate. As this type of chemistry is most commonly performed by enzymes containing pyridoxal phosphate (PLP), we focused our search on unusually distributed PLP-dependent enzymes (PLP-DEs) present in staphylococci9 including PLP-DEs from the Cys/Met metabolism family. All staphylococci contain orthologues of MetC, a cystathionine β-lyase that converts cystathionine to homocysteine as the penultimate step in methionine biosynthesis16. Previous work demonstrated that MetC from S. haemolyticus, a species in the G1 clade, does not catalyse Cys-3M3SH cleavage, suggesting another PLP-DE is responsible17. Orthologues of another PLP-DE identified in Bacillus subtilis, the putative aspartate transaminase PatA18 (Fig. 1B), are ubiquitously distributed in staphylococci. However, a second related protein, known as PatB in B. subtilis, is present in a small number of staphylococci only (Fig. 1B). In fact, the occurrence of a gene encoding this protein correlates precisely with the detection of Cys-Gly-3M3SH breakdown in our in vivo biotransformation assay (Fig. 1B). While the PatB enzymes are poorly characterised and the genes are not associated with amino acid metabolism gene clusters or operons19, the orthologues from B. subtilis (PatB) and Escherichia coli (MalY) are known to have cystathionine β-lyase activity19,20, suggesting that these enzymes might also be capable of Cys-3M3SH cleavage.

Mapping the few examples of staphylococcal PatB-like enzymes onto the global phylogeny of the Staphylococcus genus, suggests that horizontal gene transfers into staphylococcal lineages occurred on three independent occasions with the earliest being into an ancestor of G1, likely from a Bacillus-like ancestor (Fig. 2). This clade of PatB-containing staphylococci includes S. hominis, a strong producer of thioalcohol-based malodour, and its signature enzyme, which we named ShPatB, was thus studied further. Core genome analysis reveals ShPatB is a conserved core gene present in S. hominis (Supplementary Information Figure S3). To test whether ShPatB is important for malodour production, we expressed the gene in a non-malodour producing strain of S. aureus and were able to measure 3M3SH production in vivo (Supplementary Information Figure S2 D). This demonstrates that ShPatB is both necessary and sufficient for thioalcohol-based odour production in the human underarm (Fig. 1A, B).

Figure 2

Malodour producing staphylococci contain a unique C-S lyase enzyme. Maximum likelihood tree of PLP dependent C-S lyases from representative bacterial species. A unique PLP dependent PatB enzyme is found in a distinct phylogenetic clade of staphylococcal species, which we refer to as malodour producing staphylococci (coloured orange, G1). Coloured dots represent selected PLP-dependent enzymes purified for further biochemical characterisation. Orthologous PatB enzymes found in other Staphylococcusal spp. are indicated by G1, G2 and G3. Phylogenetic tree was generated using iTOL (

ShPatB is selective for branched aliphatic thioalcohol ligands

Next, we cloned and overexpressed genes encoding a representative range of PatB-type enzymes, with two MetC proteins as controls (Fig. 2, denoted by coloured dots) and purified the proteins for biochemical analysis. We compared the catalytic efficiencies (kcat/KM) of these 12 PLP-DEs for the malodour substrate Cys-3M3SH and the classical C-S lyase substrate cystathionine (Fig. 3). We observe distinct clusters of catalytic activity, with G1 PatB enzymes showing higher activity against the malodour substrate Cys-3M3SH compared to all the other PLP-DEs (Fig. 3). In particular, ShPatB exhibits selectivity towards Cys-3M3SH with a catalytic efficiency 138-fold higher that that towards cystathionine (766 M−1 min−1 and 5.53 M−1 min−1, respectively) (Fig. 3, Supplementary Information Figure S4, Table S1 and Table S2). The staphylococcal PatB-like enzymes from G2 and G3 species show very low activity towards Cys-3M3SH, suggesting that they do not contribute significantly to body odour formation given the likely micromolar concentrations of precursor present in the axilla (Fig. 3). The PatB enzymes from non-axillary microbes B. subtilis (BsPatB) and Streptococcus anginosus (SaPatB) did not discriminate between the two ligands (Fig. 3), while the enzymes from Corynebacterium jeikeium (CjAecD) and E. coli MalY (EcMalY) had higher activities against cystathionine, similar to the MetC enzymes included (ShMetC and SeMetC), which have little or no activity against Cys-3M3SH (Fig. 3 and Supplementary Information Figure S4). We also measured enzyme activity against Felinine, a close structural analog of Cys-3M3SH and a putative pheromone precursor found in cat urine21, and observed a very similar activity profile to that seen for Cys-3M3SH (Supplementary Information Figure S4, Table S3). Compared to cystathionine, the malodour precursors differ significantly in the side chains attached to the cysteine thiol, these being branched and hydrophobic, rather than linear with ionisable amino and carboxylate groups which are expected to be charged at physiological pH (Supplementary Information Figure S4). As ShPatB and G1 PatB enzymes have novel selectivity for cysteine-conjugated thioalcohol ligands, we propose that these enzymes are cysteine-thiol lyases (C-T lyases) distinct from C-S lyases acting on a broad range of cysteine-conjugated ligands (such as BsPatB and CjAecD).

Figure 3

Malodour producing staphylococci PatB enzymes are selective for Cys-3M3SH. Comparison of catalytic efficiencies (Kcat/KM) for selected PLP dependent C-S lyases against classical C-S lyase substrate l-cystathionine (y-axis) and l-Cys-3M3SH (x-axis). We show distinct clusters of activity across the PLP-DEs. MetC enzymes are selective for cystathionine only while G1 malodour-producing staphylococci are substrate selective for Cys-3M3SH, we now classify these enzymes as cysteine-thiol lyases. Other PatB orthologs (blue) display activity across both substrates whereas orthologous PatB enzymes from staphylococci (G2 & G3) show little or no activity with either substrates. Groups of staphylococcal PatB enzymes are highlighted.

Structural characterisation reveals a hydrophobic thioalcohol binding pocket

To explore the structural basis of ShPatB selectivity for these more hydrophobic malodour precursors compared to the broader C-S lyase substrate activity of BsPatB, crystal structures of the two proteins were solved and refined to resolutions of 1.6 Å (PDB ID 6QP2) and 2.3 Å (PDB ID 6QP3) respectively (Supplementary Information Figure S5A, B). ShPatB and BsPatB are homodimeric with each subunit containing a PLP moiety covalently bound to a conserved lysine residue in the catalytic site (Lys246 and Lys234 in ShPatB and BsPatB, respectively) in what is termed the internal aldimine state (Supplementary Information Figure S5 A and B). Overall, ShPatB and BsPatB are structurally conserved and belong to the type onefold of PLP-DEs22 (Supplementary Information Figure S5D). Absorption spectra indicate the presence of PLP (410 nm) covalently bound to ShPatB (Supplementary Information Figure S6A). We note, upon addition of Cys-3M3SH and additional peak at ~ 500 nm concomitant with a decrease at 410 nm (Supplementary Information Figure S6B), this species is most likely the external aldimine intermediate. A peak in this range typically indicates the presence of a PLP intermediate and is observed in cystathionine β-lyases23. After 30 s the peak reduces with a slight increase at 410 nm. As ShPatB is a β-C-S lyase we do not see any activity with l-methionine which is a γ-lyase substrate (Supplementary Information Figure S6C).

In the course of a typical PLP-DE catalysed reaction, upon substrate binding, the ε-amino group of the amino acid substrate displaces the lysine residue from the PLP to form an external aldimine24,25,26 (Supplementary Information Figure S8). The PLP and the α-amino group of the displaced lysine next facilitate electron pair and proton shuttling that lead to breakage of the C-S bond and release of 3M3SH (see Supplementary Information Figure S8 for suggested mechanism). We made several attempts to crystallise ShPatB and subsequent ShPatB catalytic mutants in the presence of the ligand Cys-3M3SH but were unable to obtain crystals suitable for X-ray structure determination. As we were unable to capture reaction intermediates by soaking ShPatB or BsPatB crystals with Cys-3M3SH, we sought insight into the mode of substrate binding by growing crystals of ShPatB in the presence of l-cycloserine (LCS). LCS is a known PLP-DE inhibitor27, that forms an external aldimine complex with PLP thereby inhibiting ShPatB (Fig. 5A, B). The structure solved at 1.4 Å (PDB ID: 6QP1) confirms the formation of the external aldimine and reveals the LCS and PLP interacting residues in the binding pocket of ShPatB (Fig. 4A, B, D and Supplementary Information Figure S5C). Mutation of key conserved PLP and LCS interacting residues reduced activity both in vitro and in vivo (Figs. 4E and 5D, E), demonstrating their important roles in binding and catalysis24. Supplementary Information Table S4 summarises the steady state kinetics for all ShPatB mutants analysed for Cys-3M3SH in vitro biotransformation. Common to PLP-DEs, a highly conserved arginine residue (Figs. 4B and 5A, C) forms an ion-pairing interaction with the carboxylate group of the amino acid moiety of the various amino acid substrates22,28,29. In ShPatB, we infer this arginine to be Arg376 (Supplementary Information Figure S5A); in the structure of the inhibitor complex, it forms a polar interaction with the C=O of LCS and is well-positioned to form a salt-bridge with the Cys-3M3SH adduct. Moreover, substitution of this residue with alanine abolishes activity both in vitro and in vivo (Figs. 4E and 5E, respectively).

Figure 4

Structural characterisation of ShPatB binding site. (A) Homodimeric structure of ShPatB. Both surface and ribbon representation are shown. (B) Zoomed view of ShPatB bound in complex with cycloserine (PDB ID 6QP1). l-cycloserine is shown in the external aldimine form bound to PLP. Coloured residues Y72 T276 denote chain A while all other residues are from chain B. (C) Modelled Cys-3M3SH complex in ShPatBLCS structure. The Cys-3M3SH ligand is modelled in the external aldimine form and docked onto the ShPatBLCS structure. Cys-3M3SH is coordinated by conserved ion pairing of its carboxylate group with the side chain of Arg376. (D) Electrostatic surface potential for ShPatBLCS and BsPatB respectively. Zoomed in views of the active site indicating the possible role of Y25 in mediating apolar interactions. In the 90 °C rotated view, we clearly see a narrow hydrophobic pocket in wild-type ShPatB whereas BsPatB lacking Y25 appears to have a more solvent accessible exposed binding site. (E) In vitro kinetics of ShPatB mutants. Mutagenesis highlights the importance of the conserved PLP interacting and ligand binding residues while revealing the importance of Y25 and E362. All structural images were generated in CCP4MG (

Figure 5

Functional analysis of ShPatB active site variants. (A) Schematic diagram of ShPatB + cycloserine complex. PLP interacting residues are indicated in yellow and ligand interacting residues are in blue. (B) Cycloserine in vitro inhibition of ShPatB with Cys-3M3SH as the substrate. End point in vitro biotransformation assay of ShPatB (2.5 μM) incubated with Cys-3M3SH (2.5 mM) in the presence or absence of cycloserine. Release of 3M3SH was labelled with DTNB and absorbance measured at 412 nm (y-axis). Cycloserine was thought to bind irreversibly to PLP27 to inactivate ShPatB however, we show that inhibition is reversed by excess PLP thus regenerating ShPatB for catalysis of Cys-3M3SH. (C) Multiple sequence alignment of C-S-β-lyases showing conserved PLP and ligand interacting residues. (D) In vivo biotransformation of Cys-3M3SH with ShPatB binding site PLP mutants and (E) ShPatB ligand interacting residues. Phylogenetic tree was generated using iTOL (

While the reaction mechanism of PLP-DEs action is well established, determining the substrate specificity of novel PLP-DEs remains a major challenge24. To gain insights into ShPatB selectivity we modelled the structure of the external aldimine form of the enzyme bound to Cys-3M3SH. The PLP adduct of Cys-3M3SH was superimposed onto equivalent atoms of the LCS external aldimine bound to ShPatB so that the α-carboxylate of the substrate forms the conserved ion-pairing interaction with Arg376. In this conformation, the side chain of Cys-3M3SH projects from the deeply recessed PLP binding pocket towards the protein surface (Fig. 4C and Supplementary Information Figure S7A and B). The aliphatic 3M3SH species fits, with minimal steric hindrance, into a spacious apolar pocket formed by the side chains of Tyr25, Val46, Tyr72, Val108, Pro134,Phe274 and Thr276 (where italics denotes a residue from the partner subunit of the dimer) (Fig. 4C). The hydrophobic character of this pocket provides few, if any, polar groups to form interactions with the side chain of cystathionine which would be expected to be zwitterionic at physiological pH (Fig. 4C, D). As a result, cystathionine binding would be accompanied by the development of unsolvated, or poorly solvated charge, lowering affinity and accounting for discrimination against this substrate as observed in our kinetic data (Fig. 3). We suggest that the hydrophobic character of this pocket accounts for the selectivity toward malodour substrates. To test this, we measured the kinetics of the ShPatB catalysed reaction with a range of cysteine-S conjugate ligands that varied in side chain length, the presence or absence of side chain branching, and side chain polarity. ShPatB clearly prefers branched aliphatic side chains followed by linear hydrophobic side groups while excluding linear charged ligands (Fig. 6B). This structural accommodation of malodour substrates represents a unique evolutionary trajectory for ShPatB not seen in other reported PatB enzymes to date.

Figure 6

ShPatB is selective for aliphatic cysteine-S-conjugates. (A) ShPatB and other G1 PatB enzymes contain unique hydrophobic residues. (B) Catalytic efficiencies (Kcat/KM) show that ShPatB has significantly higher activity for aliphatic cysteine-S-conjugate substrates compared to BsPatB. We hypothesise that unique hydrophobic residues (Tyr25 and Phe274) found only in malodour producing staphylococci mediates apolar contacts hence increased selectivity of ShPatB for these substrates. PSA indicates polar surface area calculated by BIOVA Draw 2018. (C) Structural comparison of ShPatB N-terminal region with BsPatB. BsPatB lacks equivalent Tyr25 found in ShPatB and does not provide a hydrophobic pocket. (D) ShPatB contains a highly variable region located at N-terminus. Structural sequence alignments shows a highly divergent N-terminal region between malodour producing staphylococci and orthologs. Y25 is coloured orange in malodour producing staphylococci. Red background indicates strictly conserved, red text—highly similar. The alignment was generated using MAFFT-LiNS in Jalview ( the graphic was prepared on the ESPript 3.0 server ( Phylogenetic trees were generated using iTOL (

To gain insights into this constrained substrate binding site in ShPatB, we examined a unique tyrosine residue (Tyr25) that extends into the active site in each subunit with the phenolic side chain projecting towards the PLP moiety (Figs. 4A and 6C). Tyr25 is part of a sequence divergent N-terminal region, and this residue is found only in malodour producing staphylococci (Fig. 6D). This N-terminal region is often unresolved in PLP-DE structures, and indeed, we could resolve this region only in the ShPatBLCS structure. The introduction of a Tyr25Ala substitution resulted in a sixfold increase in KM, significantly affecting Cys-3M3SH binding compared to wild-type ShPatB (Fig. 4E). We hypothesise that Tyr25 contributes to a specific hydrophobic surface in ShPatB, absent in BsPatB and other PLP-DEs (Figs. 4C and 6C), that efficiently orientates the 3M3SH moiety. In contrast to ShPatB, the BsPatB binding cavity is more solvent exposed and composed of charged residues (Fig. 4D), thus enabling the binding of polar substrates like cystathionine and the hydroxyl group of the 3M3SH moiety of Cys-3M3SH.

Supporting this, the substitution of Tyr25 to a similarly hydrophobic phenylalanine (Tyr25Phe) does not significantly affect Cys-3M3SH binding (Fig. 4E). The shape of the hydrophobic pocket is critical for Cys-3M3SH binding; mutating Thr276 to Ala resulted in an 8.5-fold increase in ShPatB KM for Cys-3M3SH (Fig. 4E). Within the neighbourhood of this apolar pocket, we observe hydrophobic residues (Tyr25, Phe274) that are unique to malodour producing staphylococci (Fig. 6A). Taken together, our observations suggest that the hydrophobic binding site in ShPatB is a key determinant of this enzyme's selectivity towards malodour-producing substrates.

Evolutionary phylogeny of malodour producing staphylococci

As noted previously (Fig. 2), the distribution of PatB enzymes among staphylococci is limited to a handful of species. In contrast, they have a much broader distribution across the Bacillus genus suggesting an ancient horizontal gene transfer (HGT) event into staphylococci. From our phylogenetic analysis, we infer this happened at least three times in staphylococci (Fig. 2), although only one of these events occurred in human associated staphylococci and led to an enzyme with high activity against Cys-3M3SH with counter selectivity against cystathionine (the G1 PatB enzymes). As the G1 enzymes are present in species that form a clear monophyletic group of staphylococci, we attempted to date the split of this clade from the other non-odour producing staphylococci to age the process of thioalcohol production. In order to determine the evolutionary phylogeny, we generated a core genome alignment of representative Staphylococcus sp. (1B). This core genome alignment was used to infer a time-scaled evolutionary phylogeny of Staphylococcus species. We used Bayesian analysis to estimate Staphylococcus species divergence time (Supplementary Information Figure S9). For the temporal scale, we used the divergence time between Staphylococcus warneri and Staphylococcus pasteuri estimated from the TimeTree database30. We show the appearance and diversification of malodour producing staphylococci from the most recent common ancestor (MRCA) approximately 60 million years ago (MYA) (95% highest posterior density (HPD) 45–89 MYA) (Fig. 7). This would imply that the emergence of this process in the staphylococcal population occurred around the same time as the early divergence of primates and the appearance of the suborder Haplorhini31. While several studies have characterised the human skin microbiome (reviewed by Grice and Segre32) relatively little is known about the composition of non-human mammals, especially using next-generation sequencing technologies. Humans have a distinct axillary microbiota that is typically less diverse compared to other primates. However, Council et al.33 showed that, in the absence of antiperspirant or deodorant usage, humans share a similar axillary microbiome to apes. They identified a core axillary microbiome dominated by Corynebacterium along with Anaerococcus, Prevotella and Staphylococcus as the most abundant taxa. While there is certainly error in this estimate of 60 MYA, we believe that the most parsimonious explanation is that this malodour producing group of staphylococci was associated with the ancestral populations of humans going back towards the divergence of primates.

Figure 7

Divergence time and evolution of Staphylococcus spp. Bayesian maximum clade credibility tree for representative Staphylococcus spp. based on core genome sequences. Branch lengths are proportional to divergence times (millions of years ago, MYA). Blue bars represent 95% highest posterior density of node age. Our data show the diversification of malodour producing staphylococci approximately 60 MYA. Phylogenetic tree was generated using FigTree V1.4.4 (

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