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1.
Chi-Pang Chuck Lin-Tat Chong Chao Chen Hak-Fun Chow David Chi-Cheong Wan Kam-Bo Wong 《PloS one》2010,5(10)
Background
The 3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.Methodology/Principal Findings
To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3'' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1'' position prefers small residues, while P2'' and P3'' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3'' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.Conclusions/Significance
Our results demonstrated a strong structure-activity relationship between the 3CLpro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors. 相似文献2.
Chunhui Liu Juzheng Sheng Juno M. Krahn Lalith Perera Yongmei Xu Po-Hung Hsieh Wenfang Dou Jian Liu Lars C. Pedersen 《The Journal of biological chemistry》2014,289(19):13407-13418
Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential
physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a
complex pathway involving multiple enzymes. In vivo regulation of this process
remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway.
Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine
5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and
specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST
position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and
Arg-190 of 2OST interact with the N-sulfo groups near the modification site,
consistent with the dependence of 2OST on N-sulfation. In contrast,
6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion
within the active site supporting the hypothesis that 2-O-sulfation occurs prior to
6-O-sulfation. Our results provide the structural evidence for understanding the
sequence of enzymatic events in this pathway. 相似文献
3.
4.
5.
Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism. 相似文献
6.
Hordeins, the natural substrates of barley (Hordeum vulgare) cysteine endoproteases (EPs), were isolated as protein bodies and degraded by purified EP-B from green barley malt. Cleavage specificity was determined by synthesizing internally quenched, fluorogenic tetrapeptide substrates of the general formula 2-aminobenzoyl-P(2)-P(1)-P(1)'-P(2)' 1-tyrosine(NO(2))-aspartate. The barley EPs preferred neutral amino acids with large aliphatic and nonpolar (leucine, valine, isoleucine, and methionine) or aromatic (phenylalanine, tyrosine, and tryptophan) side chains at P(2), and showed less specificity at P(1), although asparagine, aspartate, valine, and isoleucine were particularly unfavorable. Peptides with proline at P(1) or P(1)' were extremely poor substrates. Cleavage sites with EP-A and EP-B preferred substrate sequences are found in hordeins, their natural substrates. The substrate specificity of EP-B with synthetic peptides was used successfully to predict the cleavage sites in the C-terminal extension of barley beta-amylase. When all of the primary cleavage sites in C hordein, which occur mainly in the N- and C-terminal domains, were removed by site-directed mutagenesis, the resulting protein was degraded 112 times more slowly than wild-type C hordein. We suggest that removal of the C hordein terminal domains is necessary for unfolding of the beta-reverse turn helix of the central repeat domain, which then becomes more susceptible to proteolytic attack by EP-B. 相似文献
7.
8.
A Determinant of Substrate Specificity Predicted from the
Acyl-Acyl Carrier Protein Desaturase of Developing
Cat''s Claw
Seed 总被引:1,自引:0,他引:1
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Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase. 相似文献
9.
Catherine E. Jessop Timothy J. Tavender Rachel H. Watkins Joseph E. Chambers Neil J. Bulleid 《The Journal of biological chemistry》2009,284(4):2194-2202
The formation of disulfides within proteins entering the secretory pathway
is catalyzed by the protein disulfide isomerase family of endoplasmic
reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to
catalyze the isomerization of non-native disulfide bonds formed in
glycoproteins with unstructured disulfide-rich domains. Here we investigated
the mechanism underlying ERp57 specificity toward glycoprotein substrates and
the interdependence of ERp57 and the calnexin cycle for their correct folding.
Our results clearly show that ERp57 must be physically associated with the
calnexin cycle to catalyze isomerization reactions with most of its
substrates. In addition, some glycoproteins only require ERp57 for correct
disulfide formation if they enter the calnexin cycle. Hence, the specificity
of ER oxidoreductases is not only determined by the physical association of
enzyme and substrate but also by accessory factors, such as calnexin and
calreticulin in the case of ERp57. These conclusions suggest that the calnexin
cycle has evolved with a specialized oxidoreductase to facilitate native
disulfide formation in complex glycoproteins.The ability to form disulfide bonds within proteins entering the secretory
pathway is essential for cell survival and occurs within the endoplasmic
reticulum (ER).3 For
proteins with few disulfides, the process can be catalyzed by oxidation of
cysteine residues to form the correct, native disulfide; however, for proteins
with several disulfides, an isomerization reaction is also required to correct
non-native disulfides formed following oxidation
(1). Both these reactions are
catalyzed by a group of ER-resident proteins that belong to the protein
disulfide isomerase (PDI) family, which comprises over 17 members
(2). It is well established
that PDI and several other family members are able to catalyze the formation
and isomerization of disulfides in vitro, although the exact function
of each of the family members in vivo is unknown. It is still an open
question as to whether they all catalyze similar reactions and have distinct
substrate specificities or whether they have distinct enzymatic functions
related to the breaking and formation of disulfides.For one member of the PDI family, the function and substrate specificity is
a little clearer. ERp57 has been shown previously to interact specifically
with glycoproteins during their folding
(3). The enzyme is physically
associated with either calnexin or calreticulin
(4) and is therefore ideally
placed to catalyze correct disulfide formation within proteins entering the
calnexin/calreticulin cycle (referred to subsequently just as the calnexin
cycle). In addition, the ability of ERp57 to catalyze the refolding of
substrates in vitro is greatly enhanced if the substrate is bound to
calnexin (5). Recently,
substrates for the reduction or isomerization reaction catalyzed by ERp57 have
been identified by trapping mixed disulfides between enzyme and substrate
(6). Strikingly, there was an
overrepresentation of substrate proteins with cysteine-rich domains containing
little secondary structure, suggesting that the main function of ERp57 is in
the isomerization of non-native disulfides. ERp57 has also been shown to
function independently from the calnexin cycle. It is a component of the MHC
class I loading complex where it forms a disulfide-linked complex with tapasin
and is thought to either stabilize the complex or facilitate correct assembly
of class I molecules (7,
8). Recently, ERp57 has been
demonstrated to isomerize interchain disulfides in the major capsid protein,
VP1, of simian virus 40 (9).
The ability to dissociate VP1 pentamers by ERp57 does not require the
substrate to interact with the calnexin cycle. Hence, it is still unclear how
ERp57 recognizes its substrates, and in particular, whether this recognition
is solely determined by an interaction with the calnexin cycle.The recognition of substrates by PDI is somewhat clearer in that one
particular domain within the protein (the b′ domain) has been shown to
be primarily responsible for substrate recognition and peptide binding
(10). The corresponding domain
within ERp57 has been shown to be responsible for interaction with the
calnexin cycle (11),
suggesting that for ERp57, substrate recognition must occur outside this
domain or is determined solely by substrate interaction with calnexin via its
oligosaccharide side chain. Hence, the aim of our study was to evaluate the
necessity of the calnexin cycle both for ERp57 to recognize its substrates and
for correct folding of glycoproteins. ERp57 was found to be required for the
efficient folding of one substrate, influenza virus hemagglutinin (HA), but
only when it entered the calnexin cycle. HA did not require ERp57 to fold if
it was blocked from entering the calnexin cycle. In contrast, β1-integrin
does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked
from entering the calnexin cycle
(6). Although ERp57 may be
dispensable for the folding of some glycoproteins, the interaction with
calnexin commits them to an ERp57-dependent fate. We also found that the
majority of ERp57 substrates need to enter the calnexin cycle to be acted upon
by the enzyme, demonstrating that substrate specificity is primarily dependent
upon substrate entry into the calnexin cycle. 相似文献
10.
Andreas Naegeli Ga?lle Michaud Mario Schubert Chia-Wei Lin Christian Lizak Tamis Darbre Jean-Louis Reymond Markus Aebi 《The Journal of biological chemistry》2014,289(35):24521-24532
N-Linked protein glycosylation is a very common post-translational modification that can be found in all kingdoms of life. The classical, highly conserved pathway entails the assembly of a lipid-linked oligosaccharide and its transfer to an asparagine residue in the sequon NX(S/T) of a secreted protein by the integral membrane protein oligosaccharyltransferase. A few species in the class of γ-proteobacteria encode a cytoplasmic N-glycosylation system mediated by a soluble N-glycosyltransferase (NGT). This enzyme uses nucleotide-activated sugars to modify asparagine residues with single monosaccharides. As these enzymes are not related to oligosaccharyltransferase, NGTs constitute a novel class of N-glycosylation catalyzing enzymes. To characterize the NGT-catalyzed reaction, we developed a sensitive and quantitative in vitro assay based on HPLC separation and quantification of fluorescently labeled substrate peptides. With this assay we were able to directly quantify glycopeptide formation by Actinobacillus pleuropneumoniae NGT and determine its substrate specificities: NGT turns over a number of different sugar donor substrates and allows for activation by both UDP and GDP. Quantitative analysis of peptide substrate turnover demonstrated a strikingly similar specificity as the classical, oligosaccharyltransferase-catalyzed N-glycosylation, with NX(S/T) sequons being the optimal NGT substrates. 相似文献
11.
The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. 相似文献
12.
Benzylpenicillin acylase, which hydrolyzes benzylpenicillin to 6-aminopenicillanic acid, was found to be widely distributed among members of the Schizomycetes, particularly in gram-negative bacteria, and in the genus Nocardia. The hydrolysis of a series of biosynthetic and semisynthetic penicillins by freeze-dried cells of a strain of Nocardia and of Proteus was studied. Benzylpenicillin was the preferred substrate; all departures from the benzylpenicillin side-chain structure led to reduction of substrate activity (the greater the departure, the greater the reduction in activity). Penicillin amides and methyl esters were also hydrolyzed, as were suitable N-acyl derivatives of 7-aminocephalosporanic acid. Occurrence of an enzyme activity which hydrolyzes benzylpenicillinamide to benzylpenicillin was detected in certain strains of yeasts. 相似文献
13.
It has been shown that all selectins recognize the carbohydrate epitopes sialyl Lewisx and sialyl Lewisa. For the establishment of the structure-activity relationship, the efficient synthesis of these tetrasaccharides and derivatives is therefore of vital interest. The glycosyl transferase-mediated approach is summarized with emphasis on the use of modified acceptors and modified sugar-nucleotide donors. A survey of the involved enzymes: (1-3) and (1-4)galactosyl transferases, (2-3)sialyl transferase, FucT III and FucT VI reveals that the enzymatic synthesis is highly efficient for the rapid preparation of sialyl Lewisx- and sialyl Lewisa-derivatives. 相似文献
14.
Yoshiyuki Sakano Tsuneo Kobayashi Yoshiji Kosugi 《Bioscience, biotechnology, and biochemistry》2013,77(11):1535-1540
Yeast isoamylase was highly purified by means of salting-out with ammonium sulfate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. More than 200-fold purification was achieved through these procedures from crude yeast extract. While the purified enzyme did not attack α-1, 6-glucosidic linkages in panose, isopanose (6-malto-sylglucose), branched triose (4,6-diglucosylglucose), and isomaltosylmaltose (63-α-glucosylmaltotriose), it acted on α,β-limit dextrin to liberate glucose as well as maltose and higher oligosaccharides. Substrate specificity of the yeast isoamylase was discussed in comparison with that of plant and bacterial isoamylases (R-enzvme and pullulanase), and the mechanism of debranching of glycogen by yeast enzymes was also discussed. 相似文献
15.
Ras and Rap proteins are closely related small GTPases. Whereas Ras is
known for its role in cell proliferation and survival, Rap1 is predominantly
involved in cell adhesion and cell junction formation. Ras and Rap are
regulated by different sets of guanine nucleotide exchange factors and
GTPase-activating proteins, determining one level of specificity. In addition,
although the effector domains are highly similar, Rap and Ras interact with
largely different sets of effectors, providing a second level of specificity.
In this review, we discuss the regulatory proteins and effectors of Ras and
Rap, with a focus on those of Rap.Ras-like small G-proteins are ubiquitously expressed, conserved molecular
switches that couple extracellular signals to various cellular responses.
Different signals can activate
GEFs2 that induce the
small G-protein to switch from the inactive, GDP-bound state to the active,
GTP-bound state. This induces a conformational change that allows downstream
effector proteins to bind specifically to and be activated by the GTP-bound
protein to mediate diverse biological responses. Small G-proteins are returned
to the GDP-bound state by hydrolyzing GTP with the help of GAPs. Ras (Ha-Ras,
Ki-Ras, and N-Ras) and Rap proteins (Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C)
have similar effector-binding regions that interact predominantly with RA
domains or the structurally similar RBDs present in a variety of different
proteins. Both protein families operate in different signaling networks. For
instance, Ras is central in a network controlling cell proliferation and cell
survival, whereas Rap1 predominantly controls cell adhesion, cell junction
formation, cell secretion, and cell polarity. These different functions are
reflected in a largely different set of GEFs and GAPs. Also the downstream
effector proteins operate in a selective manner in either one of the
networks. 相似文献
16.
Masao Fujimoto Akira Kuninaka Hiroshi Yoshino 《Bioscience, biotechnology, and biochemistry》2013,77(9):1555-1561
Nuclease P1 cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split. 相似文献
17.
Martin Welin Lari Lehtiö Andreas Johansson Susanne Flodin Tomas Nyman Lionel Trésaugues Martin Hammarström Susanne Gräslund Pär Nordlund 《Journal of molecular biology》2013
Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors. 相似文献
18.
Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria 总被引:5,自引:1,他引:4
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Jennifer L. Seffernick Gilbert Johnson Michael J. Sadowsky Lawrence P. Wackett 《Applied microbiology》2000,66(10):4247-4252
Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range. 相似文献
19.
Nanne M. Kamerbeek Arjen J. J. Olsthoorn Marco W. Fraaije Dick B. Janssen 《Applied microbiology》2003,69(1):419-426
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee >99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications. 相似文献