ABSTRACT
The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.
Subject(s)
Anethum graveolens , Anethum graveolens/chemistry , Anethum graveolens/metabolism , Methyltransferases/genetics , Methyltransferases/metabolismABSTRACT
Capsaicinoids are phenolic compounds that have health benefits. However, the pungency and poor water solubility of these compounds limit their exploitation. Glycosylation is a powerful method to improve water solubility and reduce pungency while preserving bioactivity. PaGT3, a uridine diphosphate glycosyltransferase (UGT) from Phytolacca americana, is known for its ability to glycosylate capsaicinoids and other phenolic compounds. While structural information on several UGTs is available, structures of UGTs that can glycosylate a range of phenolic compounds are rare. To fill this gap, crystal structures of PaGT3 with a sugar-donor analogue (UDP-2-fluoroglucose) and the acceptors capsaicin and kaempferol were determined. PaGT3 adopts a GT-B-fold structure that is highly conserved among UGTs. However, the acceptor-binding pocket in PaGT3 is hydrophobic and large, and is surrounded by longer loops. The larger acceptor-binding pocket in PaGT3 allows the enzyme to bind a range of compounds, while the flexibility of the longer loops possibly plays a role in accommodating the acceptors in the binding pocket according to their shape and size. This structural information provides insights into the acceptor-binding mechanism in UGTs that bind multiple substrates.
Subject(s)
Glycosyltransferases , Phytolacca americana , Glycosylation , Glycosyltransferases/chemistry , Uridine Diphosphate/chemistry , Uridine Diphosphate/metabolismABSTRACT
The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.
Subject(s)
Crystallography, X-Ray/methods , Glycosyltransferases/metabolism , Molecular Docking Simulation/methods , Phytolacca americana/enzymology , Plant Proteins/metabolism , Polyphenols/metabolism , Uridine Diphosphate/metabolism , Amino Acid Sequence , Glycosylation , Glycosyltransferases/genetics , Mutation , Phylogeny , Plant Proteins/genetics , Protein Structural Elements , Substrate SpecificityABSTRACT
Uridine diphosphate glycosyltransferases (UGTs) are ubiquitous enzymes that are involved in the glycosylation of small molecules. As glycosylation improves the water solubility and stability of hydrophobic compounds, interest in the use of UGTs for the synthesis of glycosides of poorly soluble compounds is increasing. While sugar-donor recognition in UGTs is conserved with the presence of a plant secondary product glycosyltransferase (PSPG) motif, the basis of the recognition of the sugar acceptor and the regioselectivity of the products is poorly understood owing to low sequence identity around the acceptor-binding region. PaGT3, a glycosyltransferase from the plant Phytolacca americana, can glycosylate a range of acceptors. To illustrate the structure-function relationship of PaGT3, its crystal structure was determined. The sugar-donor and sugar-acceptor binding pockets in PaGT3 were recognized by comparison of its structure with those of other UGTs. The key feature of PaGT3 was the presence of longer loop regions around the hydrophobic acceptor-binding pocket, which resulted in a flexible and wider acceptor binding pocket. In this study, PaGT3 crystals were grown by co-crystallization with 18-crown-6 ether or 15-crown-5 ether. The crown-ether molecule in the asymmetric unit was observed to form a complex with a metal ion, which was coordinated on two sides by the main-chain O atoms of Glu238 from two molecules of the protein. The crown ether-metal complex resembles a molecular glue that sticks two molecules of PaGT3 together to enhance crystal growth. Thus, this result provides an insight into the substrate-recognition strategy in PaGT3 for the study of glycosyltransferases. Additionally, it is shown that crown ether-metal ion complexes can be used as a molecular glue for the crystallization of proteins.
Subject(s)
Glycosyltransferases/chemistry , Phytolacca americana/enzymology , Plant Proteins/chemistry , Crown Ethers/metabolism , Protein Conformation , Structure-Activity RelationshipABSTRACT
Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.
Subject(s)
Heme/metabolism , Iron/metabolism , Yersinia pseudotuberculosis/enzymology , Ferredoxin-NADP Reductase/metabolism , NADP/metabolism , Spectrum Analysis, Raman , Structure-Activity RelationshipABSTRACT
The stilbene compound resveratrol was glycosylated to give its 4'-O-ß-D-glucoside as the major product in addition to its 3-O-ß-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4'-O-ß-D-glucoside, and converted pinostilbene to its 4'-O-ß-D-glucoside as a major product and its 3-O-ß-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-ß-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-ß-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.
Subject(s)
Glycosides/chemical synthesis , Glycosides/pharmacology , Monophenol Monooxygenase/antagonists & inhibitors , Oxygen Radical Absorbance Capacity , Stilbenes/chemistry , Agaricales/enzymology , Biocatalysis , Chemistry Techniques, Synthetic , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glucosyltransferases/metabolism , Glycosides/chemistry , Glycosylation , ResveratrolABSTRACT
Quercetin 3-O-ß-monoglucopyranoside and quercetin 3-O-ß-maltooligosaccharide were synthesized from quercetin using glucosyltransferase-3 from Phytolacca americana and cyclodextrin glucanotransferase.
Subject(s)
Glucosides/chemistry , Glucosyltransferases/chemistry , Oligosaccharides/chemistry , Phytolacca americana/enzymology , Plant Proteins/chemistry , Quercetin/chemistry , Biocatalysis , Molecular StructureABSTRACT
Some Gram-negative pathogens utilize an extracellular heme-binding protein called hemophore to satisfy their needs for iron, a metal element essential for most living things. We report here crystal structures of heme acquisition system A from Yersinia pseudotuberculosis (HasAypt) and its Y75A mutant. The wild-type HasAypt structure revealed that the heme iron is coordinated with Tyr75 and a water molecule. The heme-bound water molecule makes extensive hydrogen bond network that includes Arg40 and Arg144 on the distal heme pocket. Arg40, highly conserved for HasAs from Yersinia species, forms a salt bridge with the propionate side chain of heme, and makes π-π stacking and hydrophobic interactions with porphyrin plane. Interestingly, similar Arg-heme interactions are also found for periplasmic heme transporter, PhuT, suggesting that this is an example of a convergent evolution and one of the important interactions for bacterial heme transportation. Heme titration, heme binding kinetics, and the crystal structures of wild-type and Y75A proteins show that, although Tyr75 is primarily important for heme capturing, other interactions with Arg40, Arg144, and hydrophobic residues also contribute for heme acquisition. We also found that HasAypt can form a dimer in solution. The structure of the domain-swapped Y75A HasAypt dimer shows the presence of two low-spin heme molecules coordinated with His84 and His140, and displacement of the Arg40 loop of dimeric Y75A HasAypt results in deformation of the heme-binding pocket. A similar rearrangement of the distal heme loop might occur in heme transfer from HasAypt to HasRypt.
Subject(s)
Arginine/metabolism , Carrier Proteins/chemistry , Heme/metabolism , Hemeproteins/chemistry , Yersinia pseudotuberculosis/chemistry , Yersinia pseudotuberculosis/metabolism , Arginine/chemistry , Crystallography, X-Ray , Heme/chemistry , Heme-Binding Proteins , Ligands , Water/chemistryABSTRACT
Resveratrol was glucosylated to its 3- and 4'-ß-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-ß-glucoside. P. americana cells converted piceatannol into its 4'-ß-glucoside. The 3- and 4'-ß-glucosides of resveratrol were further glucosylated to 3- and 4'-ß-maltosides of resveratrol, 4'-ß-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-ß-glucoside and 3-ß-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-ß-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-ß-glucoside showed high phosphodiesterase inhibitory activity.
Subject(s)
Glycosides/chemistry , Stilbenes/chemical synthesis , Stilbenes/pharmacology , Animals , Anti-Allergic Agents/chemical synthesis , Anti-Allergic Agents/chemistry , Anti-Allergic Agents/pharmacology , Biphenyl Compounds , Cell Line , Chemistry Techniques, Synthetic , Free Radical Scavengers/chemical synthesis , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Histamine Release/drug effects , Male , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Picrates , Rats , Resveratrol , Stilbenes/chemistryABSTRACT
Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.
Subject(s)
Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Iron/chemistry , Bacterial Proteins/genetics , Carrier Proteins/genetics , Heme/chemistry , Hydrophobic and Hydrophilic Interactions , Spectrum Analysis, Raman , Yersinia pseudotuberculosis/metabolismABSTRACT
The heme acquisition systemâ A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.
Subject(s)
Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Coordination Complexes/metabolism , Heme/metabolism , Iron/chemistry , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/chemistry , Binding Sites , Carrier Proteins/chemistry , Coordination Complexes/chemistry , Crystallography, X-Ray , Heme/chemistry , Indoles/chemistry , Indoles/metabolism , Iron/metabolism , Isoindoles , Mesoporphyrins/chemistry , Mesoporphyrins/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray IonizationABSTRACT
Two glucosyltransferase isozymes from Phytolacca americana, PaGT3 and PaGT2, catalyzed stereo- and regio-selective monoglucosylation of 3,5,3',4'-tetrahydroxy-trans-stilbene to yield 3,5,3',4'-tetrahydroxy-trans-stilbene-4'-O-beta-D-glucopyranoside.
Subject(s)
Glucosides/chemical synthesis , Phytolacca americana/enzymology , Stilbenes/chemical synthesis , Glucosides/biosynthesis , Glucosides/chemistry , Glucosyltransferases/metabolism , Stereoisomerism , Stilbenes/chemistryABSTRACT
A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-ß-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-ß-glucoside: 3-O-ß-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Phytolacca americana/enzymology , Phytolacca americana/genetics , Stilbenes/metabolism , Gene Expression , Glucosyltransferases/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Resveratrol , Stereoisomerism , Substrate Specificity , Time FactorsABSTRACT
Human cystathionine beta-synthase (CBS) is a pyridoxal 5'-phosphate (PLP) dependent hemoprotein, which catalyzes the condensation of serine and homocysteine. Our mutagenesis studies suggest that Arg-266 is important to sense structural changes in heme-binding site, and that Gln-222 as well as Tyr-223 are involved in interactions with substrates.
Subject(s)
Cystathionine beta-Synthase/chemistry , Cystathionine beta-Synthase/metabolism , Heme/chemistry , Heme/metabolism , Mutagenesis, Site-Directed/methods , Catalytic Domain , Cystathionine beta-Synthase/genetics , Heme/genetics , Homocysteine/metabolism , Humans , Hydrogen Sulfide/metabolism , Models, Molecular , Protein Binding , Serine/metabolism , Spectrophotometry , Structure-Activity RelationshipABSTRACT
Human cystathionine beta-synthase (CBS) catalyzes a pyridoxal 5'-phosphate (PLP) dependent beta-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 A away from heme. A possible explanation of our results is discussed on the basis of CBS structure.
Subject(s)
Cystathionine beta-Synthase/genetics , Mutation , Alanine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Catalysis , Cystathionine/genetics , Cystathionine beta-Synthase/chemistry , Cystathionine beta-Synthase/isolation & purification , Cystathionine beta-Synthase/metabolism , Heme/chemistry , Heme/genetics , Heme/metabolism , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/genetics , Pyridoxal Phosphate/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Around three million Japanese are persistently infected with HBV or HCV. Though most of them work in various industries, little is known about the actual conditions in their workplaces. To clarify the workplace conditions of workers with hepatitis, three kinds of questionnaire surveys, answered by occupational health physicians and workers with hepatitis, were carried out. The rates of workers recognized as workers with hepatitis B or C by occupational health physicians were 0.82% and 0.48% of 130,092 workers, respectively. About 30% of workers with hepatitis were engaged in "hazardous work". The percentage of workers engaged in various types of hazardous work among workers with hepatitis was nearly the same as that among all Japanese workers. About 30% of occupational health physicians witnessed exacerbation of hepatitis in the workers at their workplaces, and 22% of workers with hepatitis experienced exacerbation of hepatitis. The rate of workers with hepatitis who had experienced exacerbation was not significantly different between workers with and without hazardous work. Workers with hepatitis have strong concerns about the relationship between work and exacerbation. As causes of exacerbation, occupational health physicians cited "unknown", "drinking" and "quit treatment" while workers with hepatitis answered "work-related causes", besides "unknown" and "drinking."
Subject(s)
Hepatitis B, Chronic/epidemiology , Hepatitis C, Chronic/epidemiology , Occupational Health/statistics & numerical data , Occupations , Adult , Alcohol Drinking , Female , Health Knowledge, Attitudes, Practice , Health Surveys , Humans , Industry , Japan/epidemiology , Male , Middle Aged , Prevalence , WorkplaceABSTRACT
In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.
Subject(s)
Myoglobin/chemistry , Myoglobin/genetics , Amino Acid Substitution , Amino Acids/chemistry , Amino Acids/genetics , Amino Acids/metabolism , Animals , Benzothiazoles , Binding Sites , Catalysis , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Heme/chemistry , Heme/genetics , Hydrogen Peroxide/chemistry , Kinetics , Models, Molecular , Myoglobin/metabolism , Oxidation-Reduction , Peroxidases/chemistry , Peroxidases/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry/methods , Stereoisomerism , Sulfides/chemistry , Sulfides/metabolism , Sulfonic Acids/chemistry , Sulfonic Acids/metabolism , WhalesABSTRACT
The H64D/V68A and H64D/V68S mutants of Myoglobin are found to oxidize thioanisole with high enantioselectivity and reactivity. These mutants are also capable of enantioselective binding of alpha-methylbenzylamine, which mimics an expected sulfoxidation intermediate. The kinetic study of the amine binding shows that the Fe-O bond cleavage in the intermediate may be the chiral discrimination step of the sulfoxidation.