A novel family of bacterial dioxygenases that catalyse the hydroxylation of free L-amino acids

L-isoleucine-4-hydroxylase (IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. L-methionine, L-leucine, L-isoleucine and L-threonine were found to be catalysed by the investigated enzymes, producing L-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free L-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed.     

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.     

Oxidative biotransformation of thioanisole by Rhodococcus rhodochrous IEGM 66 cells

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0-1.5 g/l) concentrations of sulfide substrate.     

The analysis and application of a recombinant monooxygenase library as a biocatalyst for the Baeyer-Villiger reaction

Because of their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the chemoenzymatic synthesis of a broad range of useful compounds. In this study, we investigated the microbial Baeyer-Villiger oxidation and sulfoxidation of thioanisole and bicyclo[3.2.0]hept-2-en-6-one using whole Escherichia coli cells that recombined with each of the Baeyer-Villiger monooxygenases originated from Pseudomonas aeruginosa PAO1 and two from Streptomyces coelicolor A3(2). The three BVMOs were identified in the microbial genome database by a recently described protein sequence motif; e.g., BVMO motif (FXGXXXHXXXW). The reaction products were identified as (R)-/(S)sulfoxide and 2-oxabicyclo/3-oxabicyclo[3.3.0]oct-6-en-2-one by GC-MS analysis. Consequently, this study demonstrated that the three enzymes can indeed catalyze the Baeyer-Villiger reaction as a biocatalyst, and effective annotation tools can be efficiently exploited as a source of novel BVMOs.     

l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

l-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of l-leucine and l-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of l-methionine and l-ethionine in the same manner as previously described l-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.     

Terpene ligands as the basis of catalytic systems for the asymmetric oxidation of phenylphenacyl sulfide

Terpene ligands (1S,2S,5S)-3-[{2-[(2-hydroxybenzylidene)amino]ethyl}imino]-2,6,6-trimethylbicyclo[3.1.1.]heptane-2-ol and 3-({2-[(2-hydroxy-2,6,6-trimethylbicyclo[3.1.1.]hept-3-ilidene)amino]ethyl}imino)-2,6,6-trimethylbicyclo[3.1.1.]heptane-2-ol have been synthesized for the first time. The efficiency of complexes based on terpene and salen ligands in asymmetric sulfoxidation has been compared. Catalytic systems based on terpene ligands have been used for the first time in the asymmetric oxidation of phenylphenacyl sulfide with the formation of sulfoxide with an enantiomeric excess of 47%.     

Fenugreekine,a new steroidal sapogenin-peptide ester of Trigonella foenum-graecum

A new C₂₇-steroidal sapogenin-peptide ester, fenugreekine, has been isolated from seeds of Trigonella foenum-graecum. On acid hydrolysis, it afforded diosgenin, yamogenin, (25R)-spirosta-3,5-diene, a mixture of three isomeric (2S,3R,4R-, 2S,3R,4S-, 2S,3S,4R-)-4-hydroxyisoleucine lactones, 4′-hydroxyisoleucyl-4-hydroxyisoleucine lactone, and a C₁₄-dipeptide which was partially characterized. On the basis of this chemical transformation and spectral (UV, IR, PMR, MS) evidence of fenugreekine and its transformation products, the steroidal sapogenin-peptide ester is assigned structure (1). The two dipeptides also have not been encountered before in nature or prepared synthetically. The compound shows a number of interesting pharmacological and virological activities.     

A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine: identification of an enzyme possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase activity

A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and alpha-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, alpha-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.     

A novel L-isoleucine metabolism in Bacillus thuringiensis generating (2S,3R,4S)-4-hydroxyisoleucine, a potential insulinotropic and anti-obesity amino acid

4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for L-isoleucine (L-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2S,3R,4S)-HIL. The (2S,3R,4S)-HIL was oxidized to (2S,3R)-AMKP by a NAD(+)-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B(12) antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP.     

Oxidative biotransformation of thioanisole by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> IEGM 66 cells

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0–1.5 g/l) concentrations of sulfide substrate.     

Enhancement of activity of lipase-displaying yeast cells and their application to optical resolution of (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate

Rhizopus oryzae lipase (ROL) was displayed on the cell surface of Saccharomyces cerevisiae via the Flo1 N-terminal region (1100 amino acids), which corresponds to a flocculation functional domain. The activity of lipase-displaying yeast whole-cell biocatalysts was enhanced 7.3-fold by incubation of the yeast cells at 20 degrees C in distilled water for 8 days after 8 day cultivation. The amount of lipase molecules present in cell wall and intracellular fractions was found to be increased 4.5- and 1.8-fold, respectively, by incubation, which proves that ROL molecules are expressed during incubation. The ROL-displaying yeast whole-cell biocatalyst with enhanced activity was successfully catalyzed by optical resolution of the pharmaceutical precursor (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate. Moreover, it showed stable activity through at least eight reaction cycles. These results demonstrate that ROL-displaying yeast cells with enhanced activity by incubation in distilled water are very effective in industrial bioconversion processes.     

Regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

The regio- and stereospecific oxidation of fluorene, dibenzofuran, and dibenzothiophene was examined with mutant and recombinant strains expressing naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. The initial oxidation products were isolated and identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. Salicylate-induced cells of Pseudomonas sp. strain 9816/11 and isopropyl-beta-D-thiogalactopyranoside-induced cells of Escherichia coli JM109(DE3)(pDTG141) oxidized fluorene to (+)-(3S,4R)-cis-3,4-dihydroxy-3,4-dihydrofluorene (80 to 90% relative yield; > 95% enantiomeric excess [ee]) and 9-fluorenol (< 10% yield). The same cells oxidized dibenzofuran to (1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzofuran (60 to 70% yield; > 95% ee) and (3S,4R)-cis-3, 4-dihydroxy-3,4-dihydrodibenzofuran (30 to 40% yield; > 95% ee). Induced cells of both strains, as well as the purified dioxygenase, also oxidized dibenzothiophene to (+)-(1R,2S)-cis-1,2-dihydroxy-1, 2-dihydrodibenzothiophene (84 to 87% yield; > 95% ee) and dibenzothiophene sulfoxide (< 15% yield). The major reaction catalyzed by naphthalene dioxygenase with each substrate was stereospecific dihydroxylation in which the cis-dihydrodiols were of identical regiochemistry and of R configuration at the benzylic center adjacent to the bridgehead carbon atom. The regiospecific oxidation of dibenzofuran differed from that of the other substrates in that a significant amount of the minor cis-3,4-dihydrodiol regioisomer was formed. The results indicate that although the absolute stereochemistry of the cis-diene diols was the same, the nature of the bridging atom or heteroatom influenced the regiospecificity of the reactions catalyzed by naphthalene dioxygenase.     

Disposition kinetics of ML-1035 sulfoxide enantiomers and the prochiral sulfide in rats

ML-1035, 4-amino-5-chloro-2-[2-(methylsulfinyl)ethoxy]-N-[2-(diethylamino)ethyl]benzamide, is a sulfoxide compound and a racemic gastroprokinetic agent with a chiral center at the sulfur atom. We have investigated the disposition kinetics of (R)-ML-1035 sulfoxide (R) and (S)-ML-1035 sulfoxide (S) after the single enantiomers and the racemic mixture were administered to rats in separate experiments. There was no noticeable chiral inversion after either enantiomer dose. Both enantiomers were rapidly absorbed. After dosing with enantiomers or with the racemate, the resulting plasma concentration-time curve of R was closely parallel to that of S in both intravenous and oral experiments, suggesting that the two enantiomers have approximately the same disposition kinetics. After intravenous enantiomer doses, only S underwent conversion to sulfide, suggesting that sulfidation in the liver is enantioselective. However, the enantioselective sulfidation after intravenous dosing did not introduce a difference in the global plasma disposition profiles between R and S, since the reduction reaction is a minor metabolic process. Other metabolic reactions such as sulfonation and mono-N-desethylations were not enantioselective. After oral administration, conversion to sulfide was observed for both enantioners, implicating the existence of a nonhepatic pathway in sulfidation. Administration of a prochiral sulfide dose was associated with an enantioselective sulfoxidation, in which the R/S concentration ratios increased as a function of time. In addition, enantiomeric interaction causing changes in pharmacokinetic parameters was observed after the oral racemate dose, while the interaction is negligible after an intravenous racemate dose, indicating a route dependency in enantiomeric interaction. © 1993 Wiley-Liss, Inc.     

Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans.

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.     

Preparation of chiral derivatives of beta-Ala containing the alpha-phenylethyl group: useful starting materials for the asymmetric synthesis of beta-amino acids

The use of microwave (MW) irradiation for the condensation reaction between acetophenone and alpha-phenylethylamine to prepare (R,R)-bis[alpha-phenylethyl]amine results in significantly reduced reaction times relative to the use of conventional heating. In this protocol, a secondary amine, (R,R)-bis(alpha-phenylethyl)amine is treated with acryloyl chloride to afford conjugated amide N,N-bis[(R)-alpha-phenylethyl]prop-2-enamide, (R,R)-3. 1,4-Addition of alpha-phenylethylamine to unsaturated (R,R)-3 affords propanamide N,N-Bis[(R)-alpha-phenylethyl]-3-N-[(S)-alpha-phenylethyl]amino-propanamide, (R,R,S)-4, which can be alkylated with high diastereoselectivity to give derivative N,N-Bis[(R)-alpha-phenylethyl]-3-N'-[(S)-alpha-phenylethyl]amino-propanamide, (R,R,S,S)-5. Hydrogenolysis of (R,R,S,S)-5 catalyzed by palladium hydroxide and final hydrolysis (4 N HCl) resulting in the formation of (S)-alpha-benzyl-beta-alanine, (S)-7, is facilitated by MW irradiation. The use of MW irradiation in this step prevents racemization of the desired amino acid. The present protocol constitutes one of the simplest strategies for the asymmetric synthesis of biologically relevant alpha-substituted-beta-amino acids since it takes advantage of inexpensive, commercially available beta-Ala and either (R)- or (S)-alpha-phenylethylamine as chiral auxiliary. The required time for this protocol is approximately 90 h, which can be carried out in 5 d.     

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.     

Hydroperoxide-dependent sulfoxidation catalyzed by soybean microsomes

The sulfoxidation of methiocarb, an aromatic-alkyl sulfide pesticide, catalyzed by soybean microsomes was found to be strongly stimulated in the presence of cumene and linoleic acid hydroperoxides. We have shown that this S-oxidation, which does not require cofactors such as NAD(P)H, is an hydroperoxide-dependent reaction: 18O2-labeling experiments demonstrated that the oxygen atom incorporated into the sulfoxide originated from hydroperoxides rather than from molecular oxygen. In the absence of exogenous hydroperoxides, soybean microsomes catalyzed methiocarb sulfoxide formation at a basal rate dependent on their endogenous hydroperoxides, especially those derived from free fatty acids. The nature of the sulfoxidase is discussed. Our results seem to rule out the participation of cytochrome P-450 in this oxidation, whereas the studied sulfoxidase presents some similarities to plant peroxygenase.     

Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.     

A double mutation in the extracellular Ca2+-sensing receptor's venus flytrap domain that selectively disables L-amino acid sensing

The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.