Identification of odoriferous sulfanylalkanols in human axilla secretions and their formation through cleavage of cysteine precursors by a C-S lyase isolated from axilla bacteria

Human axillary odor is known to be formed upon the action of Corynebacteria sp. on per se odorless axilla secretions. Besides the known odoriferous acids, we report the occurrence in human axilla secretions of four odoriferous sulfanylalkanols, namely 3-sulfanylhexan-1-ol (3), 2-methyl-3-sulfanylbutan-1-ol (4), 3-sulfanylpentan-1-ol (5), and 3-methyl-3-sulfanylhexan-1-ol (6). These compounds have a pungent sweat/kitchen odor, also reminiscent of onions with some fruity connotations, and perception thresholds in the pg/l range. It was postulated that the odorless precursors for these compounds are cysteine conjugates. Bacterial isolates obtained from the human axilla and belonging to the Corynebacteria were, indeed, found to have the enzymatic capacity to release various thiols from cysteine conjugates. The metC gene, which is known to code for a cystathione-beta-lyase, was cloned from the axilla isolate Corynebacterium striatum Ax20 and heterologously expressed in E. coli. The pure recombinant enzyme cleaves various cysteine conjugates and has a similar substrate specificity as the cell homogenates of the wild-type. The recombinant enzyme was finally incubated with odorless axilla secretions and shown to release odoriferous thiols.     

Identification of the precursor of (S)-3-methyl-3-sulfanylhexan-1-ol, the sulfury malodour of human axilla sweat

A careful study of human axillary microflora led us to the identification of a new strain of Staphylococcus haemolyticus. The role in axillary malodour formation of this microorganism was compared to those of Corynebacterium xerosis and Staphylococcus epidermidis, upon incubation on sterile human eccrine and apocrine axilla sweat. St. haemolyticus was responsible for the strongest sulfury malodour and the generation of the volatile sulfur compound (VSC) (S)-3-methyl-3-sulfanylhexan-1-ol (3). In this study, we investigated the nonvolatile precursors of VSCs. Human axillary sweat was collected, fractionated and analysed by HPLC/APCI-MS (High-Pressure Liquid Chromatography coupled to Atmospheric Pressure Chemical Ionisation Mass Spectrometry). The precursor of 3 was identified as [1-(2-hydroxyethyl)-1-methylbutyl]-L-cysteinylglycine (Cys-Gly-(S)-conjugate; 12). Because Cys-Gly-(S)-conjugates are key intermediates in the glutathione biodetoxification pathway, other derivatives of 12, specifically glutathione-(S)-conjugate 11 and Cys-(S)-conjugate 13, were prepared. Compounds 11 and 13 were not detected by HPLC/MS of sterile sweat. Synthetic homologues 11, 12, and 13 were incubated with C. xerosis, St. heamolyticus, and St. epidermidis. We observed efficient conversion of precursors 12 and 13 to form VSCs when incubated with St. haemolyticus, with a clear preference for 12. C. xerosis and St. epidermidis were less efficient in cleaving Cys-Gly-(S)-conjugate 12 to form the corresponding thiol 3. Incubation of glutathione-(S)-conjugate 11 never led to the formation of 3 under the experimental conditions employed.     

3-Methyl-3-sulfanylhexan-1-ol as a major descriptor for the human axilla-sweat odour profile

This study sets out to redress the lack of knowledge in the area of volatile sulfur compounds (VSCs) in axillary sweat malodour. Sterile odourless underarm sweat (500 ml) was collected from 30 male volunteers after excessive sweating. Five strains of bacteria, Corynebacterium tuberculostearicum, Corynebacterium minutissimum, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Bacillus licheniformis, were isolated and characterised for their ability to generate an authentic axillary odour from the sweat material collected. As expected, all of the five bacterial strains produced strong sweat odours. Surprisingly, after extensive olfactive evaluation, the strain of Staphylococcus haemolyticus produced the most sulfury sweat character. This strain was then chosen as the change agent for the 500 ml of odourless underarm sweat collected. After bacterial incubation, the 500-ml sample was further processed for GC-olfactometry (GC-O), GC/MS analysis. GC-O of an extract free of organic acids provided three zones of interest. The first was chicken-sulfury, the second zone was onion-like, and the third zone was sweat, clary sage-like. From the third zone, a new impact molecule, (R)- or (S)-3-methyl-3-sulfanylhexan-1-ol, was isolated and identified by GC/MS, MD-GC, and GC AED (atomic emission detector). (S)-3-methyl-3-sulfanylhexan-1-ol was sniff-evaluated upon elution from a chiral GC column and was described as sweat and onion-like; its opposite enantiomer, (R)-3-methyl-3-sulfanylhexan-1-ol, was described as fruity and grapefruit-like. The (S)-form was found to be the major enantiomer (75%).     

A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla

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 Zn(2+)-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 effects of modulation of gamma-glutamyl transpeptidase activity in HepG2 cells on thiol homeostasis and caspase-3-activity

The present studies aimed to elucidate how the modulation of gamma-glutamyl transpeptidase (gammaGT) activity in human hepatoma (HepG2) cell line influences H(2)O(2) production, caspase 3 activity, protein S-thiolation by glutathione (GSH), cysteinyl-glycine (Cys-Gly) and cysteine (Cys), and the level of other redox forms of these thiols. The experiments showed that 1-h stimulation of gammaGT elevated H(2)O(2) production, leading to prooxidant conditions. After 24-h stimulation, H(2)O(2) concentration was at the control level, while Cys-Gly-, Cys- and GSH-dependent S-thiolation was markedly increased, which was accompanied by a drop in caspase-3 activity. The inhibition of gammaGT activity by acivicin led to H(2)O(2) decrease after 1-h incubation which still persisted after 24 h. The inhibition of gammaGT activity in HepG2 cells was also connected with the lowering of S-thiolation with Cys and Cys-Gly and with increasing of caspase-3 activity. The results of our studies indicate that the modulation of gammaGT activity can be used to change cellular redox status, and can affect Cys- and Cys-Gly-dependent S-thiolation and caspase-3 activity. We suggest that the role of high gammaGT activity in HepG2 cells can be connected with production of reactive oxygen species and with S-thiolation with Cys and Cys-Gly that can influence activity of caspase 3.     

Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis

We report characterization of SUPERROOT1 (SUR1) as the C-S lyase in glucosinolate biosynthesis. This is evidenced by selective metabolite profiling of sur1, which is completely devoid of aliphatic and indole glucosinolates. Furthermore, following in vivo feeding with radiolabeled p-hydroxyphenylacetaldoxime to the sur1 mutant, the corresponding C-S lyase substrate accumulated. C-S lyase activity of recombinant SUR1 heterologously expressed in Escherichia coli was demonstrated using the C-S lyase substrate djenkolic acid. The abolishment of glucosinolates in sur1 indicates that the SUR1 function is not redundant and thus SUR1 constitutes a single gene family. This suggests that the "high-auxin" phenotype of sur1 is caused by accumulation of endogenous C-S lyase substrates as well as aldoximes, including indole-3-acetaldoxime (IAOx) that is channeled into the main auxin indole-3-acetic acid (IAA). Thereby, the cause of the "high-auxin" phenotype of sur1 mutant resembles that of two other "high-auxin" mutants, superroot2 (sur2) and yucca1. Our findings provide important insight to the critical role IAOx plays in auxin homeostasis as a key branching point between primary and secondary metabolism, and define a framework for further dissection of auxin biosynthesis.     

Combinatorial synthesis by nature: volatile organic sulfur-containing constituents of Ruta chalepensis L

Ongoing interest in discovering new natural fragrance and flavor ingredients prompted us to examine a solvent extract of sulfurous-sweaty smelling Ruta chalepensis L. (Rutaceae) plant material more closely. Twenty-one sulfur-containing constituents of similar structures were identified by GC/MS techniques. Amongst them, 14 have never been described to occur in nature. The compounds 1-18 belong to a family of natural flavor and fragrance molecules having a 1,3-positioned O,S moiety in common. The identities of the natural constituents were confirmed by comparison with synthetic reference samples, and the organoleptic properties of the latter were studied. The relative and absolute configurations of the four stereoisomers of 4-methyl-3-sulfanylhexan-1-ol (5) were established by stereoselective synthesis. The natural isomers consisted of a 65 : 35 mixture of (3R,4S)-5 and (3S,4S)-5.     

Insulin stimulates the release of a subset of GPI-anchored proteins in a G-protein independent manner

The glycosyl-phosphatidylinositol anchored protein, membrane dipeptidase (EC 3.4.13.19) is released from the surface of 3T3-L1 adipocytes in response to insulin treatment through the action of a phospholipase C. The present study investigates the role of guanine-nucleotide binding proteins (G-proteins) in this process. Treatment of permeabilized 3T3-L1 adipocytes with GTP gamma S did not cause release of membrane dipeptidase into the medium, while GDP beta S did not inhibit the insulin-stimulated release of membrane dipeptidase. Other activators of G-proteins, including the tetradecapeptide mastoparan, pertussis toxin and AlF, also caused no significant release of membrane dipeptidase from the surface of the 3T3-L1 adipocytes. From these observations it is concluded that G-proteins are not involved in the insulin stimulated release of membrane dipeptidase. Although X-Pro aminopept idase (EC 3.4.11.9) is GPI-anchored in 3T3-L1 adipocytes as shown by digestion with bacterial phosphatidylinositol specific phospholipase C, it was not released upon insulin treatment of the cells, indicating that only a subset of the GPI-anchored proteins are susceptible to insulin-stimulated release.     

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

Glutathione-degrading enzymes of microvillus membranes

Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.     

C-S lyase activities in leaves of crucifers and non-crucifers, and the characterization of three classes of C-S lyase activities from oilseed rape (Brassica napus L.)

C-S lyases in plants are involved in primary and secondary metabolism, and in glucosinolate-containing species may be involved in glucosinolate biosynthesis. Extracts from oilseed rape ( Brassica napus ) leaves were assayed for several C-S lyase activities. Four activities [using L -cystine, L -cystathionine, S -(2-benzothiazolyl)- L -Cys (SBC) and S -benzyl- L -Cys] were investigated in detail. All are developmentally regulated (highest in youngest leaves), and differentially inhibited by iodoacetamide, N -ethylmaleimide (NEM) and ethylenediaminetetraacetic acid (EDTA). Thermal stabilities and pH optima were also distinct. Competitive inhibition of the SBC lyase activity with a variety of sulphur-containing compounds indicated that cystine lyase contributes to SBC degradation, and this enzyme may cleave a wide range of compounds, both aliphatic and aromatic, but other 'SBC lyases' were also present. Putative aromatic glucosinolate intermediates were cleaved by the rape enzymes. Developmental and biochemical studies indicate that at least three classes of C-S lyase activity are present in rape leaves: cystathionine β -lyase, cystine lyase and a group of relatively non-specific lyases. C-S lyase preparations from other glucosinolate- and non-glucosinolate-containing species were capable of cleaving a number of aliphatic and aromatic conjugates. The highest activities were detected in glucosinolate-containing species and Allium cepa (onion). C-S lyase activities in non-glucosinolate-containing species (tobacco, Nicotiana tabacum, and barley, Hordeum vulgare ) were much lower.     

Dug1p Is a Cys-Gly Peptidase of the ��-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

GSH metabolism in yeast is carried out by the γ-glutamyl cycle as well as by the DUG complex. One of the last steps in the γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the constitutent amino acids. Saccharomyces cerevisiae extracts carry Cys-Gly dipeptidase activity, but the corresponding gene has not yet been identified. We describe the isolation and characterization of a novel Cys-Gly dipeptidase, encoded by the DUG1 gene. Dug1p had previously been identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an alternate GSH degradation pathway and has also been suggested to function as a possible di- or tripeptidase based on genetic studies. We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. This activity requires zinc or manganese ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly. Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We also show that the Dug1p Schizosaccharomyces pombe orthologue functions as the exclusive Cys-Gly peptidase in this organism. The human orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by complementation of the dug1Δ mutant and by biochemical characterization, which revealed a high substrate specificity and affinity for Cys-Gly. The results indicate that the Dug1p family represents a novel class of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) present in almost all eukaryotes (barring a few protozoa) and in a few prokaryotes (1). In the cell, glutathione exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the millimolar range), a relatively low redox potential (-240 mV), and a high stability conferred by the unusual peptidase-resistant γ-glutamyl bond are three of the properties endowing GSH with the attribute of an important cellular redox buffer. GSH also contributes to the scavenging of free radicals and peroxides, the chelation of heavy metals, such as cadmium, the detoxification of xenobiotics, the transport of amino acids, and the regulation of enzyme activities through glutathionylation and serves as a source of sulfur and nitrogen under starvation conditions (2, 3). GSH metabolism is carried out by the γ-glutamyl cycle, which coordinates its biosynthesis, transport, and degradation. The six-step cycle is schematically depicted in Fig. 1 (2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism. γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two steps in glutathione biosynthesis. γ-glutamyltranspeptidase, γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are involved in glutathione catabolism. Activities responsible for γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and 5-oxoprolinase activities have not been detected, which has led to the suggestion of the presence of an incomplete, truncated form of the γ-glutamyl cycle (4) made of γ-glutamyl transpeptidase (γGT)⁴ and Cys-Gly dipeptidase and only serving a GSH catabolic function. Although γGT and Cys-Gly dipeptidase activities were detected in S. cerevisiae cell extracts, only the γGT gene (ECM38) has been identified so far. Cys-Gly dipeptidase activity has been identified in humans (5, 6), rats (7–10), pigs (11, 12), Escherichia coli (13, 14), and other organisms (15, 16), and most of them belong to the M17 or the M1 and M19 metallopeptidases gene families (17).S. cerevisiae has an alternative γGT-independent GSH degradation pathway (18) made of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex. Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its Schizosaccharomyces pombe homologue. We also show that the mammalian orthologue of DUG1, CNDP2, can complement the defective utilization of Cys-Gly as sulfur source of an S. cerevisiae strain lacking DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase activity in vitro, with a strong preference for Cys-Gly over all other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the metallopeptidases M20A family and have been known to carry carnosine (β-alanyl-histidine) and carnosine-like (homocarnosine and anserine) peptidase activity (20, 21). This study thus reveals that the metallopeptidase M20A family represents a novel Cys-Gly peptidase family, since only members of the M19, M1, and M17 family were known to carry this function.     

Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction

Isoprene formation in a rat liver cytosolic fraction is shown to be increased 146-fold by acid treatment. This acid catalysis is dependent upon prior incubation of the cytosolic fraction with DL-mevalonate and is stimulated when the incubation also contains ATP. Formation of isoprene proceeds linearly through 5 h of acid treatment and is nearly complete at 10 h. These results suggest that the acid-catalyzed isoprene formation arises from the decomposition of dimethylallyl pyrophosphate via a carbonium ion mechanism. Chemical model studies using 3-methyl-2-buten-1-ol and 3-methyl-3-buten-1-ol (the alcohols corresponding to dimethylallyl pyrophosphate and isopentenyl pyrophosphate, respectively) confirm this hypothesis. At a pH less than or equal to 1, an 85% decomposition of 3-methyl-2-buten-1-ol to isoprene occurred after 24 h, while 3% of 3-methyl-3-buten-1-ol was converted to isoprene under identical conditions and time. It is concluded that the predominant immediate precursor of isoprene is dimethylallyl pyrophosphate and at low pH the ultimate fate of dimethylallyl pyrophosphate is complete conversion to isoprene. These conclusions have important biochemical and methodological implications.     

The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine.

S-(beta-Aminoethyl)-cysteine (AEC) resistance was achieved in Corynebacterium glutamicum by cloning a chromosomal 1.5-kb EcoRV-BglII DNA fragment on a multicopy plasmid. DNA sequence analysis of the 1.5-kb DNA fragment revealed an open reading frame (ORF326) which represents the AEC resistance gene, designated aecD. The aecD gene directs the synthesis of a 36-kDa protein which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aecD gene is a nonessential gene and mediates AEC resistance only in an amplified state. C. glutamicum strains harboring an amplified aecD gene can utilize AEC as an alternative nitrogen source, indicating that the AEC resistance mechanism is due to AEC degradation. Since the AEC degradation products analyzed by high-pressure liquid chromatography were found to be pyruvate and aminoethanethiol (cysteamine), it was concluded that the aecD gene encodes a C-S lyase with alpha, beta-elimination activity. Besides AEC, the C-S lyase was also able to use cysteine, cystine, and cystathionine as substrates.     

Insulin stimulates the release of a subset of GPI-anchored proteins in a G-protein independent manner

The glycosyl-phosphatidylinositol anchored protein, membrane dipeptidase (EC 3.4.13.19) is released from the surface of 3T3-L1 adipocytes in response to insulin treatment through the action of a phospholipase C. The present study investigates the role of guanine-nucleotide binding proteins (G-proteins) in this process. Treatment of permeabilized 3T3-L1 adipocytes with GTPgammaS did not cause release of membrane dipeptidase into the medium, while GDPbetaS did not inhibit the insulin-stimulated release of membrane dipeptidase. Other activators of G-proteins, including the tetradecapeptide mastoparan, pertussis toxin and AlF3, also caused no significant release of membrane dipeptidase from the surface of the 3T3-L1 adipocytes. From these observations it is concluded that G-proteins are not involved in the insulin-stimulated release of membrane dipeptidase. Although X-Pro aminopeptidase (EC 3.4.11.9) is GPI-anchored in 3T3-L1 adipocytes as shown by digestion with bacterial phosphatidylinositol-specific phospholipase C, it was not released upon insulin treatment of the cells, indicating that only a subset of the GPI-anchored proteins are susceptible to insulin-stimulated release.     

Conformational analysis of cysteine-containing peptides and prospects of their application to 213Bi chelating in antitumor therapy

The method of conformational analysis was applied to the spatial structures of peptide analogues of phytochelatins and some fragments of metallothioneins: (Cys-Gly)₃, (Cys-Gly)₃-Asp, (Cys-Gly)₃-Glu, (Cys-βAla)₃, (Cys-γGlu)₃, and (Cys-Gly-Gly)₃. All the possible low-energy conformations of the molecules were revealed and the role of intra-and interresidual interactions in the formation of their spatial structures was determined. A different tendency of the molecules under study for acceptance of conformations favorable for binding bismuth ions was shown. Low-energy structures providing an optimum binding of bismuth ion were shown to be most frequent for (Cys-βAla)₃ peptide. Among the analogues of peptide fragments of the metallothioneins, lacking in natural peptides, low-energy pentapeptide CCXXC fragments (where X = Gln, Asn, Phe, Tyr, or Gly) were revealed. In the α-helical conformations of these pentapeptides, the distance between the sulfur atoms corresponds to that in Bi₂S₃.     

Genetic variation in the odorant receptor OR2J3 is associated with the ability to detect the "grassy" smelling odor, cis-3-hexen-1-ol

The ability to detect many odors varies among individuals; however, the contribution of genotype to this variation has been assessed for relatively few compounds. We have identified a genetic basis for the ability to detect the flavor compound cis-3-hexen-1-ol. This compound is typically described as "green grassy" or the smell of "cut grass," with variation in the ability to detect it linked to single nucleotide polymorphisms (SNPs) in a region on human chromosome 6 containing 25 odorant receptor genes. We have sequenced the coding regions of all 25 receptors across an ethnically mixed population of 52 individuals and identified 147 sequence variants. We tested these for association with cis-3-hexen-1-ol detection thresholds and found 3 strongly associated SNPs, including one found in a functional odorant receptor (rs28757581 in OR2J3). In vitro assays of 13 odorant receptors from the region identified 3 receptors that could respond to cis-3-hexen-1-ol, including OR2J3. This gene contained 5 predicted haplotypes across the 52 individuals. We tested all 5 haplotypes in vitro and several amino acid substitutions on their own, such as rs28757581 (T113A). Two amino acid substitutions, T113A and R226Q, impaired the ability of OR2J3 to respond to cis-3-hexen-1-ol, and together these two substitutions effectively abolished the response to the compound. The haplotype of OR2J3 containing both T113A and R226Q explains 26.4% of the variation in cis-3-hexen-1-ol detection in our study cohort. Further research is required to examine whether OR2J3 haplotypes explain variation in perceived flavor experience and the consumption of foods containing cis-3-hexen-1-ol.