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Applied and Environmental Microbiology, June 2002, p. 2716-2725, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2716-2725.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Streptomyces Serine Protease (DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of 1,4-Dihydropyridine Calcium Antagonists

Akira Arisawa,1* Motoko Matsufuji,1 Takashi Nakashima,1 Kazuyuki Dobashi,1 Kunio Isshiki,1 Takeo Yoshioka,1 Shigeru Yamada,2 Haruo Momose,2 and Seiichi Taguchi2,3*

Bioresource Laboratories, Mercian Corporation, Fujisawa, Kanagawa 251-0057,1 Department of Biological Science and Technology, Science University of Tokyo, Noda-shi, Chiba 278,,2 School of Agriculture, Meiji University, Kawasaki-shi, Kanagawa 214-8571, Japan3

Received 10 December 2001/ Accepted 20 March 2002


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ABSTRACT
 
Streptomyces viridosporus A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (dhpA) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of Streptomyces subtilisin inhibitor (SSI), from Streptomyces albogriseolus. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of dhpA resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent S. viridosporus A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.


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INTRODUCTION
 
1,4-Dihydropyridines (4-aryl-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylates) are known to have potent antihypertensive and vasodilative actions through calcium antagonism (5, 31). Various 1,4-dihydropyridine derivatives have been developed for clinical purposes and are used as drugs against hypertension and ischemic heart disease. Although the two enantiomers possessing an asymmetric carbon at the position 4 have been reported to have different biological activities (14, 20, 30), most 1,4-dihydropyridines are provided as racemates. Single enantiomers of dihydropyridines [in most cases, the (4S)-enantiomers] have higher therapeutic activity than the other enantiomers. The first chiral 1,4-dihydropyridine drug was introduced clinically in 1992. The clinical importance of and demand for chiral 1,4-dihydropyridine drugs have been increasing steadily over the past few years because of the growing awareness that the individual isomers of drugs having a chiral center should be evaluated in detail in the process of drug development. Chiral 1,4-dihydropyridines can be obtained by the following methods: (i) chemical resolution of racemate forms (14), (ii) enantioselective chemical synthesis (13), and (iii) enzymatic hydrolysis or transesterification of prochiral diesters (1, 4, 7-9, 11). The enzymatic method is very likely to be more advantageous than the other methods because it involves simple processes with both high selectivity and high yield.

Two research groups have independently reported enantioselective hydrolysis and enantioselective transesterification of prochiral 1,4-dihydropyridines by proteases (1, 7, 8). It has also been reported that enantioselective hydrolysis occurs with lipases when prochiral 1,4-dihydropyridines containing acyloxymethyl groups at the C-3 and C-5 positions are used as substrates (9, 11). The resulting chiral half esters of the 1,4-dihydropyridines produced by the proteases or the lipases, regardless of which chirality they have, can be key intermediates for production of (4R)-1,4-dihydropyridine drugs such as Barnidipine (8, 30, 32) and Dexniguldipine (18). All enzymes used in the previous studies were commercially available. It is of great interest to investigate whether there are novel enzymes more suitable for this kind of reaction. This prompted us to screen for microorganisms producing such enzymes, which would be of considerable value to the drug industry.

In this report, we describe (i) isolation of two actinomycete strains that can produce (4R) half esters from prochiral 1,4-dihydropyridine diesters; (ii) cloning of the gene responsible for an enzyme (DHP-A) from one of the strains, Streptomyces viridosporus A-914; (iii) enhanced expression of the enzyme in heterologous hosts and in the parent strain; and (iv) comparative biochemical studies with homologous enzymes of protease substrate preference and inhibitory regulation by endogenous proteinaceous protease inhibitors.


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MATERIALS AND METHODS
 
Genetic manipulations, chemicals, and enzymes.
Genetic manipulation for Streptomyces strains and Escherichia coli (e.g., isolation of total DNA, transformation, plasmid isolation, colony hybridization, PCR, and DNA sequencing) were performed according to the standard protocols described by Hopwood et al. (12) and Sambrook et al. (19), respectively. Restriction enzymes and T4 DNA ligase were purchased from Takara (Kyoto, Japan). Protease P6, a serine protease from Aspergillus melleus, was obtained from Amano Enzyme Inc. (Nagoya, Japan). All 1,4-dihydropyridines used as substrates and standards in this study (M-801, P-902, and enantiomers of M-802 and P-903) (Fig. 1) were originally synthesized and, if necessary, separated via diastereomeric salts in our laboratory (20). All synthetic peptide substrates to test for substrate preference were purchased from Sigma Chemical Co.



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  		FIG. 1. Enantioselective hydrolysis of the prochiral 1,4-dihydropyridine diesters to chiral (4R) half esters.

Screening.
Culture stocks of actinomycetes and fungi from our original culture collections were used for screening. The microorganisms to be screened were cultivated in 2 ml of ISP-2 medium (for actinomycetes) (comprising 0.4% glucose, 0.4% yeast extract, and 1% malt extract [pH 7.3]) or in 2 ml of FI medium (for fungi) (comprising 2% potato starch, 2% soybean meal, 1% glucose, 0.1% KH2PO4, 0.05% MgSO4 · 7H2O, 0.05% Adecanol LG109 [Asahi Denka Kogyo, Tokyo, Japan]) in a test tube at 28°C for 3 days with constant shaking at 220 rpm. A 20-µl portion of a dimethyl sulfoxide solution of M-801 (100 mg/ml) was added to each growing culture, and the cultivation was continued for 20 h. After the pH of the culture was adjusted to 4.0 with 1 N HCl, M-801 and its hydrolyzed products were extracted with 1 ml of ethylacetate. The extracts were evaporated to dryness. The residues were dissolved in 100 µl of ethylacetate. An aliquot (10 µl) of each resulting solution was spotted on thin-layer chromatography (TLC) plates. The plates were then developed with solvent composed of CHCl3-methanol (6:1). Hydrolysis of M-801 (Rf, 0.59) to M-802 (Rf, 0.53) was checked by visual inspection of the TLC plates under the UV light (254 nm) or by iodine pigmentation, if necessary.

Cultivation of strains and biotransformation of 1,4-dihydropyridines.
Streptomyces strains were grown for 4 days at 28°C in C medium (2% glucose, 2% soluble starch, 2% soybean meal, 0.5% yeast extract, 0.25% NaCl, 0.32% CaCl2 · 2H2O, 5 µg of FeSO4 · 7H2O per ml, 5 µg of MnSO4 · 5H2O per ml, 5 µg of ZnSO4 · 7 H2O per ml, pH 7.4) with shaking at 220 rpm. Fungal strains were grown at 28°C for 3 days in FI medium at the same shaking rate. The culture was centrifuged at 3,000 x g for 10 min at 4°C. A 0.5-ml portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 µg of M-801 per ml) in a test tube. The mixture was incubated at 40°C for 3 to 24 h.

HPLC analysis of biotransformation products.
After the pH of the reaction mixture was adjusted to 3.0 with 1 N HCl, the reaction mixture was extracted with an equal volume of ethylacetate. A 200-µl portion of the ethylacetate layer was then evaporated to dryness. The residual pellet was dissolved in 500 µl of the mobile phase used in the subsequent high-performance liquid chromatography (HPLC) (20 mM KH2PO4-methanol, 1:1). This sample solution (20 µl) was applied to an HPLC system equipped with a YMC-pack ODS-A column (150 mm by 4.6 mm [inside diameter]); YMC Co., Ltd., Kyoto, Japan). The column was developed at 50°C with 20 mM KH2PO4-methanol (1:1) at a flow rate of 0.8 ml/min. M-801 (retention time, 5.3 min) and its monoester (M-802; retention time, 4.4 min) were detected by UV absorption at 350 nm. P-902 (retention time, 7.0 min) and its monoester (P-903; retention time, 5.1 min) were measured under the same HPLC conditions except that the mobile phase was 60% (in water) methanol-acetic acid (1,000:1) and the column temperature was 35°C. The amount of each product was quantitated from the peak area of HPLC based on that of corresponding standard.

Enantioselective analysis.
To determine the chirality of M-802 by enantioselective chromatography, we used an ULTRON ES-OVM column (150 by 4.6 mm [inside diameter]; Shinwa Chemical Industries, Ltd., Kyoto, Japan). The sample solution (20 µl), prepared after a 24-h reaction, was applied to the column, which was developed at 50°C with 0.02 M KH2PO4-2-propanol (9:1) at a flow rate of 1.0 ml/min. The enantiomers were detected by UV absorption at 350 nm. The chirality of P-903 was determined under the same HPLC conditions except that the mobile phase was 0.02 M KH2PO4-2-propanol (8:2). The retention times of the (4R)- and (4S)-P-903 enantiomers were 14 to 15 min and 16 to 17 min, respectively.

Purification of the enzyme that catalyzes enantioselective hydrolysis (DHP-A).
S. viridosporus A-914 was grown in 1 liter of C medium at 28°C for 4 days in a 3-liter jar fermentor with an aeration rate of 0.5 vol/vol/min. Cells were removed from the growing culture by centrifugation (8,000 x g) for 20 min at 4°C. Ammonium sulfate was added to the supernatant liquid slowly with stirring to yield a 60% saturated solution, and the solution was continuously stirred for 3 h at 4°C. After removal of the precipitate by centrifugation (9,000 x g) for 20 min at 4°C, additional ammonium sulfate was added, yielding an 80% saturated solution, and this solution was stirred for 15 h at 4°C. The precipitate was collected by centrifugation (9,000 x g) for 20 min at 4°C and dissolved in 20 ml of distilled water. This solution was applied to Butyl-Toyopearl 650 column (275 by 26 mm [inside diameter]; TOHSO, Tokyo, Japan) that had been equilibrated with 50 mM Tris-HCl (pH 7.5) containing 1.8 M ammonium sulfate. The column was washed with the equilibrating buffer, and then proteins were eluted with a 2,000-ml linear gradient of 1.8 to 0.4 M ammonium sulfate in the same buffer. Fractions (12 ml each) were collected at a flow rate of 49 ml/h. The active fractions eluted at around 0.7 M ammonium sulfate were collected together and desalted by filtration using an Ultrafree membrane (Millipore). The final enzyme solution (~10 ml) was dispensed in small aliquots and stored at -80°C until use. The enzyme concentration was determined using a protein assay kit (Bio-Rad catalog no. 500-0001).

Tests for protease activity, lipase activity, and enzyme inhibition.
A mixture of DHP-A solution (50 µl of 0- to 1-mg/ml solutions) and 2 mM potassium acetate (150 µl) was added to 1 ml of a substrate solution (20 mM Tris-HCl, 10 mM NaOH, 6 mg of Hammarsten casein per ml [pH 8.5]). The resulting mixture was incubated at 30°C for 10 min. This reaction was stopped by addition of 1 ml of trichloroacetic acid solution (110 mM trichloroacetic acid, 220 mM sodium acetate, 330 mM acetic acid) and centrifuged at 8,000 x g for 5 min. Protease activity for casein was determined by measuring the absorbance at 275 nm of the supernatant liquid. A DHP-A solution (200 µg/ml) was also tested for lipase activity, using a Lipase UV Autotest kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). To examine enzyme inhibition by protease inhibitors, 400 µl of 250 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] (pH 7.5) was mixed with 600 µl of either culture supernatant of S. viridosporus A-914 or protease P6 solution (3 mg/ml). Phenylmethylsulfonyl fluoride (PMSF) (300 µM) or chymostatin (80 µM) was added to the mixture. M-801 was then added to a final concentration of 300 µg/ml. The resulting mixture was incubated at 30°C for 1 h. The inhibition of the enantioselective hydrolysis was measured from the productivity of M-802 determined by HPLC analysis as described above.

Cloning of an S. viridosporus A-914 gene (dhpA) responsible for enantioselective hydrolysis
The genomic DNA isolated from S. viridosporus A-914 was partially digested with Sau3AI, and electrophoresed in a 0.8% agarose gel. DNA fragments of 3 to 9 kb in length were recovered from the excised gel with an Ultrafree C3 HV disposable microcentrifuge filtration unit (Millipore). The genomic library of S. viridosporus A-914 was constructed according to standard protocols (12), using pIJ702 (15) as a vector and Streptomyces lividans TK24 as a host strain (12). The DNA fragments were inserted into the unique BglII site of pIJ702 with T4 DNA ligase. S. lividans TK24 was transformed with the ligation mixture. After drug resistance selection (using 50 µg of thiostrepton per ml) on R2YE plates (12), the transformants were patch cultured on tryptic soy broth (Difco)-1.5% agar plates containing M-801 (500 µg/ml) at 28°C for 5 days. Agar blocks (0.1 cm3/block) around colonies were excised, placed on lanes divided by scraped parallel lines on TLC plates, and dried thoroughly to peel off the TLC plates. TLC was performed as described under "Screening" above. A transformant that showed hydrolysis activity for M-801 was isolated, and its harboring plasmid (pDE88) was prepared.

Determination of the complete sequence of the dhpA gene.
Genomic DNA from S. viridosporus A-914 (100 µg) was partially digested with Sau3AI to generate fragments ranging from 40 to 50 kb in length. A cosmid library was prepared using pHC79 (Boehringer Mannheim) (10) as a vector. The ligation mixture was packaged using Gigapack II Gold (Stratagene) and transduced in E. coli XL1-Blue MR (Stratagene). The resulting library of 1,000 colonies was plated on a 10- by 90-mm Luria-Bertani agar plate. The colonies were transferred to a nylon membrane Hybond-N+ (Amersham Biosciences) and lysed with a denaturing solution (0.5 M NaOH, 1.5 M NaCl). The DNA was immobilized on the membrane by baking at 80°C for 2 h. Colony hybridization was performed using a 0.2-kb DNA fragment as a probe, which was prepared by amplifying a region from nucleotide 2341 to 2439 by PCR. A positive cosmid was isolated and termed pDE22. Since this clone did not cover the end of the gene but went to position 3577, subsequent library screening was performed using a 0.56-kb fragment as a probe, which was amplified from a region ranging from position 3000 to 3560. In this screening, a clone which gave a positive hybridization signal was isolated and termed pDE72. The complete sequence of dhpA was determined by assembling the sequence data from pDE88, pDE22, and pDE72.

PAGE.
S. viridosporus A-914 and its recombinant strain with pDE88 were grown at 28°C for 4 days in tryptic soy broth with shaking at 220 rpm. Other strains were grown at 28°C for 3 days in the same medium with shaking at 220 rpm. Thiostrepton (25 µg/ml) was added to the medium to grow the recombinant strains. The culture supernatant of each strain (1.0 ml) was concentrated by microcentrifuge filtration using an Ultrafree-MC centrifugal filter device PL-10 (Millipore). The samples were adjusted to 100 µl by adding distilled water. These sample solutions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (16). In the test for complex formation of proteases with inhibitor proteins, native PAGE was performed with a 10% polyacrylamide gel. In the preparation of sample solutions before loading onto the gel, heat treatment and addition of both SDS and reducing reagent (2-mercaptoethanol) were omitted. Protein bands in the gel were stained with 2% (wt/vol) Coomassie brilliant blue R-250.

Enzyme assay.
Streptomyces strains and their recombinant strains were grown in C medium. For the recombinant strains, 25 µg of thiostrepton per ml was added to the medium for selective culture conditions. After cultivation at 28°C for 3 to 4 days, each of the cultures was centrifuged at 3,000 x g for 10 min at room temperature. A 500-µl portion of the supernatant liquid was added to an equal volume of an assay premix (200 mM Tris-HCl [pH 8.0], 1,000 mM NaCl, 600 µg of M-801 per ml) in a test tube. The mixture was incubated at 40°C for 30 min. The reaction was stopped by diluting 10-fold with a solution comprised of 20 mM KH2PO4 and methanol (1:1). A 200-µl portion of the diluted reaction mixture was then transferred to a microcentrifuge tube and centrifuged at 10,000 x g for 5 min. The supernatant liquid (20 µl) was analyzed by HPLC under the conditions described under "HPLC analysis of biotransformation products" above. The specific activity of the enantioselective hydrolysis from each strain was measured based on the definition that 1 U of enzyme hydrolyzes 1 nmol of M-801 to 1 nmol of (4R)-M-802 per min. Conversion rates were calculated from values of HPLC peak areas of M-801 and (4R)-M-802 after the reaction.

Purification and sequence determination of SIL23.
A proteinaceous protease inhibitor, termed Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein 23 (SIL23), was purified to homogeneity from the culture supernatant of S. viridosporus A-914 as described previously (27). Automated sequence analysis of the purified SIL23 was carried out by Edman degradation with Applied Biosystems model 476A and 473A protein sequencers.

Protease substrate preference analysis.
Chromogenic synthetic peptides were used as substrates to test for differences in substrate preference between DHP-A and SAM-P45. Each hydrolysis reaction was carried out at 25°C in a 1.0-ml reaction mixture containing 100 nM enzyme, 100 µM substrate, 100 mM Tris-HCl (pH 8.5), 0.1% dimethyl sulfoxide, and 10 mM CaCl2. The specific activity of the enzyme toward each substrate was determined by measuring the amount of p-nitroanilide released after proteolytic cleavage at 410 nm.

Nucleotide sequence accession number.
The nucleotide sequence data reported here (for dhpA) appear in the GenBank database under accession number AB007809.


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RESULTS AND DISCUSSION
 
Screening of microbial strains producing an enzyme that hydrolyzes 1,4-dihydropyridines enantioselectively.
We developed a rapid assay method to detect 1,4-dihydropyridine hydrolysis activity and screened microorganisms with high hydrolyzing activities from our culture collections, including approximately 5,000 actinomycete strains and 1,000 fungal strains. Of the two available 1,4-dihydropyridine substrates (M-801 and P-902), M-801 was used for the screening (Fig. 1). No background reaction was observed in the control medium, in which no strains were grown. Many actinomycete strains (more than 40%) had some degree of activity that could hydrolyze M-801 to its monocarboxy (M-802) and/or dicarboxy derivatives. The hydrolyzing activity was difficult to detect in some strains because of degradation of the substrate, presumably through an unknown anabolic pathway(s). During this screening, markedly higher activities forming M-802 were found in the culture broths of two streptomycete strains, S. viridosporus A-914 and Streptomyces clavus N-1284, and two fungal strains, Paecilomyces sp. strain FI-1007 and Botryodioplodia sp. strain FI-741.

The product generated by the action of the culture broth from S. viridosporus A-914 was identified as (4R)-M-802 by HPLC analyses (Fig. 2). Significantly, no production of (4S)-M-802 was detected, indicating that this hydrolysis was strictly enantioselective. The other strains (S. clavus N-1284 and the two fungal strains) also gave (4R)-M-802 alone (data not shown). A similar enzyme assay using P-902 as a substrate revealed that all of the selected strains hydrolyzed P-902 to (4R)-P-903 (data not shown). Further biochemical and application studies were continued for S. viridosporus A-914, since this strain showed the highest and the most stable activity.



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  		FIG. 2. HPLC analyses of a 1,4-dihydropyridine half ester, M-802, formed by the enzyme from S. viridosporus A-914 and determination of chirality. M-801 was incubated in the culture supernatant of S. viridosporus A-914 for 3 to 24 h. (a and b) Samples before the reaction (a) and after reaction for 3 h (b) were analyzed by HPLC. Under the conditions used, it is known that the retention times of M-801 and M-802 are 5.3 and 4.4 min, respectively. (c and d) To determine the chirality of the half ester (M-802) produced after the hydrolysis, chiral HPLC analysis was applied to authentic racemic M-802 (c) and to the product after the complete reaction (24 h) (d). The retention time of (4R)-M802 was 5.3 min, while that of (4S)-M802 was 6.4 min. mABs, milli-absorbance units.

Purification and characterization of the enzyme that catalyzes enantioselective hydrolysis of 1,4-dihydropyridines.
The enantioselectively hydrolytic enzyme from S. viridosporus A-914 that is capable of converting M-801 to (4R)-M-802 was termed DHP-A. To characterize DHP-A in detail, the enzyme was purified from the culture supernatant of S. viridosporus A-914. After ammonium sulfate precipitation followed by hydrophobic interaction chromatography, 10 mg of the electrophoretically homogeneous enzyme was finally obtained from 1 liter of the culture. The N-terminal amino acid sequence of DHP-A was determined to be Leu-Asp-Thr-Ser-Val-Gly. The molecular mass, optimum pH, optimum temperature, and other properties of the purified DHP-A are summarized in Table 1. Similarly, the properties of protease P6, a commercially available serine protease from A. melleus that is known to have the same hydrolyzing activity for 1,4-dihydropyridines, were also examined as a reference. Compared with protease P6, DHP-A was more alkaliphilic and more thermostable during the course of the enantioselective hydrolysis. Like protease P6, DHP-A was reactive not only toward 1,4-dihydropyridines but also toward natural protease substrates such as casein. PMSF and chymostatin effectively inhibited the 1,4-dihydropyridine-hydrolyzing and proteolytic activities of DHP-A, indicating that DHP-A can be classified as a serine protease in which an active-site serine residue is critical for both reactions.


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  		TABLE 1. Properties of enantioselectively hydrolyzing enzyme (DHP-A) from S. viridosporus A-914 and protease P6

Next, we compared the abilities of DHP-A and protease P6 to generate the chiral 1,4-dihydropyridine half esters in a batch reaction test. P-902, a 1,4-dihydropyridine diester that is more water soluble than M-801 and thus more suitable for industrial mass production, was used as a substrate. We could add P-902 at a high concentration (10 mg/ml) to the reactor so that the concentration of the substrate was not a limiting factor. The concentrations of enzymes were set to 2 mg/ml for DHP-A and 3 mg/ml for protease P6, which represent equal initial activities based on the productivity during 15 min. As seen in Fig. 3, the production of (4R)-P-903 (a half ester of interest) by protease P6 diminished almost completely in 8 h, probably because the enzyme activity became inactivated, whereas the DHP-A-catalyzed hydrolysis proceeded with little loss of enzyme activity during the reaction. At the end of the reaction (13.5 h), the production of the half ester P-903 with 2 mg of DHP-A per ml was 2.8 times as much as that with 3 mg of protease P6 per ml. Productivity with 1 mg of DHP-A per ml (equivalent to half of the initial activity of protease P6 at 3 mg/ml) exceeded that with 3 mg of protease P6 per ml after 9 h and was finally 1.7-fold greater. It was concluded that DHP-A would be more suitable for the hydrolysis than protease P6 in terms of enzyme activity and durability, resulting from higher stability to temperature and pH and possibly for unnatural substrates.



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  		FIG. 3. Time courses of the production of (4R)-1,4-dihydropyridine half ester P-903 by DHP-A and protease P6. P-902 (final concentration of 10 mg/ml) was reacted with DHP-A at 2 mg/ml (•) or 1 mg/ml ({circ}) or with protease P6 at 3 mg/ml ({square}) in 250 mM sodium phosphate buffer adjusted to pH 9.0 (optimal for DHP-A) or to pH 8.0 (optimal for protease P6) at 40°C.

Cloning of the gene encoding the 1,4-dihydropyridine-hydrolyzing enzyme (dhpA) and its homologous and heterologous expression.
To clone the gene encoding DHP-A, S. lividans TK24, which has no hydrolyzing activity for 1,4-dihydropyridines, was used as a host strain to construct a genomic library of S. viridosporus A-914. Using the TLC assay, we isolated a transformant strain of S. lividans TK24, exhibiting the 1,4-dihydropyridine hydrolyzing activity, from approximately 1,000 thiostrepton-resistant transformants. Conversion of M-801 to (4R)-M-802 by this transformant showed the same enantioselectivity as that by the parent strain, strongly suggesting that the gene for the 1,4-dihydropyridine-hydrolyzing enzyme was present in the recombinant plasmid. The plasmid isolated from the transformant, pDE88 (Fig. 4A), had a 2.9-kb Sau3AI (a)-Sau3AI (b) insert from the genomic DNA of S. viridosporus A-914, presumably containing the gene of interest. Deletion of a 0.64-kb SacI (a)-SacI (b) fragment from pDE88 did not decrease the hydrolyzing activity, whereas deletion of a 1.79-kb MluI (a)-MluI (b) fragment caused loss of the activity (data not shown). This indicates that the gene of interest was located within the 2.54-kb SacI (b)-Sau3AI (b) region of the insert.



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  		FIG. 4. Overproduction of DHP-A in the original and heterologous host strains. (A) Structure of pDE88. Typical restriction enzyme recognition sites and locations of the genes contained in the pIJ702-based plasmid are shown. Only two of several Sau3AI sites, which are involved in the cloning site, are shown. The thiostrepton resistance gene (tsr) (12) and the tyrosinase gene (mel) (2) are indicated by a thick open arrow and a thick solid arrow, respectively. An open box indicates the 2.9-kb Sau3AI (a)-Sau3AI (b) partially digested fragment cloned from S. viridosporus A-914. The coding region and orientation of dhpA are shown by a thin solid arrow along the open box. The ORF upstream of the tyrosinase gene (ORF438) (2), which is divided into two parts by insertion of the dhpA fragment at the unique BglII site, is indicated by a stippled box. (B) SDS-PAGE of total proteins in the culture supernatants from the DHP-A-producing Streptomyces strains. Lane 1, protein size marker; lane 2, plasmid-free S. lividans TK24; lane 3, S. lividans TK24(pDE88); lane 4, plasmid-free S. clavus N-1284, lane 5, S. clavus N-1284(pDE88); lane 6, S. viridosporus A-914; lane 7, S. viridosporus A-914(pDE88); lane 8, purified authentic DHP-A (4 µg).

With regard to the industrial use of DHP-A, it is very important to obtain strains with improved productivity of the enzyme. The dhpA-carrying plasmid pDE88, constructed from a high-copy vector (pIJ702), was introduced into the parent strain of S. viridosporus A-914 and also into S. clavus N-1284, which produces another 1,4-dihydropyridine-hydrolyzing enzyme. The obtained recombinants, as well as S. lividans TK24(pDE88), showed stable retention of the plasmid. To examine enzyme productivity, total proteins from the culture supernatants of the three recombinant strains were analyzed by SDS-PAGE (Fig. 4B). In both S. lividans(pDE88) and S. clavus(pDE88), expression of a considerable amount of DHP-A was observed as a product of 53 kDa. The indigenous hydrolyzing enzyme from S. clavus N-1284, tentatively called DHP-N, is not yet well characterized. Preliminary data showed that the mature form of DHP-N has a molecular mass of 45 kDa. The amount of DHP-A produced by S. viridosporus(pDE88) was clearly elevated over the basal level observed in the parent S. viridosporus A-914.

We evaluated the specific activity of 1,4-dihydropyridine enantioselective hydrolysis in the culture supernatants of the three recombinant strains. Consistent with the results of SDS-PAGE analysis, the recombinant strains exhibited 2.4- to 4.2-fold increased enzyme activity (per milliliter of culture supernatant) compared with the parent strain S. viridosporus A-914 (Table 2). In S. clavus(pDE88), the enhanced productivity resulted from a combination of two different enzymes, the host-indigenous hydrolyzing enzyme (DHP-N) and the heterologously expressed DHP-A, both of which have the same catalytic activity. These data suggest that these strains could be promising as sources of a low-cost DHP-A applicable to the industrial field, although there is still some room for optimization of enzymatic functions. In the future, directed evolution might be a powerful way to increase the activity and stability of DHP-A, in light of several studies in which serine proteases with improved functions have been created by this technology with relatively simple screening methods (17, 24).


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  		TABLE 2. Enantioselective hydrolysis activity in recombinant strains

Sequence analysis of the dhpA gene.
Sequence analysis of the 2.54-kb SacI(b)-Sau3AI(b) region of pDE88 revealed the presence of only one long open reading frame (ORF) whose 3' end was truncated, thus resulting in readthrough to the noncoding strand of ORF438 (the upstream ORF of the tyrosinase gene [2]) in the pIJ702 vector. The consequent ORF tentatively encodes 823 amino acids (aa) with a molecular mass of 83.5 kDa by artificial fusion at the C-terminal portion with the polypeptide (89 aa; molecular mass, 8.9 kDa) from the ORF438 region. A putative promoter sequence, containing a -35 region (TTGAGG; nucleotide positions 208 to 213) and a -10 region (CATACT; nucleotide positions 232 to 237) similar to the streptomycete consensus promoter sequence (21), was found about 100 bp upstream of one of the possible start codons (GTG at nucleotide position 338) in the ORF. The N-terminal amino acid sequence determined for the purified DHP-A (Leu-Asp-Thr-Ser-Val-Gly) was found at positions 1 to 6 in the corresponding amino acid sequence. Therefore, we concluded that this ORF codes for DHP-A. A comparison of the N-terminal amino acid sequence with the deduced amino acid sequence, together with the fact that DHP-A is an extracellular enzyme, led us to conclude that this enzyme possesses a pre-pro sequence for secretion and maturation.

To determine the full coding sequence of dhpA, we isolated two cosmid clones (pDE22 and pDE72) from the S. viridosporus genomic library after colony hybridization using 3' regions of dhpA as probes. Sequence analysis of the two clones enabled the determination of the complete sequence of dhpA. The length of the complete dhpA was 3,315 bp, encoding 1,105 aa (residues -204 to 901) with a molecular mass of 114.1 kDa. This massive gene product would be processed at the C-terminal end as well as at the N-terminal end and would result in the mature enzyme of 53 kDa found in the culture supernatant of S. viridosporus A-914. The N-terminal residue of the mature enzyme was determined to be Leu. Therefore, the N-terminal portion of the unprocessed DHP-A, comprising 204 aa of 21.1 kDa, would be removed by the processing. However, the C-terminal processing (probably at around aa 545) that leads to the 53-kDa mature form of DHP-A has not yet been analyzed precisely.

The entire amino acid sequence of DHP-A exhibited high homology (70.8% identity) to SAM-P45 (22), which was identified by us as a subtilisin-like protease in Streptomyces albogriseolus. As can be seen in Fig. 5, the mature region and the C-terminal half of the C-terminal prodomain (residues 690 to 901) of DHP-A showed higher homology with the corresponding region of SAM-P45 (88.1 and 77.8% identity, respectively), whereas the N-terminal half of the C-terminal prodomain (residues 414 to 689) showed less homology (43.5% identity). The low-homology region would be responsible for the variation in the C-terminal processing site that causes the difference in the molecular masses of the mature forms (DHP-A, 53 kDa; SAM-P45, 45 kDa). Although the function of the C-terminal prodomain in DHP-A is still unclear, it was reported for SAM-P45 that the domain might be involved in membrane anchoring (22). SAM-P45 is also known as a target protease toward the proteinaceous inhibitor protein SSI (6), and it is thought to have roles in secondary metabolism or cellular morphogenesis (22). This protease family is probably common in streptomycetes (data not shown), and a widespread distribution of this type of enzyme would account for why many streptomycete strains were found to have the hydrolyzing activity for M-801 in the screening. Potential catalytic triad residues that are common in subtilisin-like proteases (3) are conserved at positions Asp29, His61, and Ser238 (Fig. 5A), confirming the conclusion from its inhibitor sensitivities that DHP-A is a serine protease (Table 1).



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  		FIG. 5. Amino acid sequence comparison of the mature regions (A) and C-terminal pro regions (B) of DHP-A and SAM-P45. Identical amino acid residues are indicated by outlined letters. Amino acid residues of the conserved catalytic triad (Asp29, His61, and Ser238) are indicated by solid arrowheads.

Comparison of protease substrate preferences of DHP-A and SAM-P45.
Because of the sequence similarity between DHP-A and SAM-P45, it was of interest to investigate their enzymatic properties, in particular, their protease substrate preferences. The preference of DHP-A for synthetic substrates was examined with a series of chromogenic peptide substrates possessing Lys, Arg, Phe, Leu, Ala, or Glu at the P1 site, as listed in Table 3. A similar tendency in substrate preference (preference for the basic amino acids) was observed for DHP-A and SAM-P45, as expected from their high structural homology. Also, SAM-P45 was found to have an effective hydrolyzing activity toward M-801 (but less than that of DHP-A) by TLC and HPLC analysis (data not shown).


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  		TABLE 3. Substrate preferences of DHP-A and SAM-P45

In vitro complex formation of DHP-A with inhibitor proteins.
We previously found that proteinaceous protease inhibitors homologous to SSI (6) are widely distributed in various streptomycetes, and we termed them SIL proteins (25, 28). The discovery of these "natural mutants" of SSI prompted us to explore the presence of this type of inhibitor protein in S. viridosporus A-914. From the analogy to the presence of an endogenous inhibitor (SSI) of SAM-P45 (22, 29), the presence of an endogenous inhibitor of DHP-A was predicted. By use of the same purification strategy adopted for the other SIL proteins (27, 28), a 13-kDa proteinaceous protease inhibitor was purified to homogeneity with respect to inhibitory activity against subtilisin BPN'. The sequence of the N-terminal amino acids of the protein (Tyr-Ala-Pro-Ser-Ala-Leu-Val-Phe-Thr-Leu-Gly-His-Gly-Glu-Glu-Ala-Ala-Val-Thr-Thr-Val) was very similar to that of the N-terminal amino acids of SSI (Asp-Ala-Pro-Ser-Ala-Leu-Tyr-Ala-Pro-Ser-Ala-Leu-Val-Leu-Thr-Val-Gly-Lys-Gly-Val-Ser-Ala-Thr-Thr-Ala-Ala-Pro) (identical amino acid residues are in boldface). Thus, this inhibitor appears to be a member of SIL protein family and was termed SIL23. By size exclusion chromatography, SIL23 was found to exist in a homodimeric form, as do the other SIL proteins (data not shown). To determine whether SIL23 is a potent target protease inhibitor of DHP-A, the interaction of SIL-23 with DHP-A was analyzed by native PAGE. DHP-A was mixed with SIL23 (5:1 molar excess of inhibitor) for 30 min. As shown in Fig. 6, a band corresponding to that expected for the complexed form of DHP-A and SIL23 was detected in the upper part of the gel above free noncomplexed SIL23. This suggested that the hydrolyzing activity of DHP-A would be inhibited by the binding of SIL23. Cross associations of SIL23 and SSI with the heterologous proteases (SIL23 versus SAM-P45 and SSI versus DHP-A) were also detected, indicating the functional similarity between these two inhibitors.



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  		FIG. 6. In vitro complex formation of enzymes (DHP-A and SAM-P45) and inhibitors (SIL23 and SSI) revealed by native PAGE analysis. Enzymes were mixed with inhibitors in a molar ratio of approximately 1:5 (enzyme to inhibitor subunit) in the buffer solution (100 mM Tris-HCl [pH 8.5], 10 mM CaCl2) and then subjected to native PAGE. The arrowheads indicate the protein bands corresponding to complexes of enzymes with inhibitors.

The inhibitory profiles of the four combinations of two enzymes with two inhibitors (DHP-A with SIL23, DHP-A with SSI, SAM-P45 with SSI, and SAM-P45 with SIL23) were examined by measuring residual proteolytic activities of the enzymes in the presence of SIL23 or SSI at various inhibitor ratios. The binding of DHP-A and SIL23 was weakest among the four combinations (data not shown). This may account for the selective screening of DHP-A from among the many streptomycete sources. In fact, three SSI target proteases, including SAM-P45 were identified in an SSI-deficient Streptomyces mutant strain (23, 26, 29). Similarly, a mutation causing SIL23 deficiency in S. viridosporus would be effective in the production of a large amount of free DHP-A.

Inhibitory regulation of the hydrolysis of DHP-A.
To clarify the inhibition of the hydrolyzing activity of DHP-A by the endogenous inhibitor SIL23 or by the exogenous related inhibitor SSI, the enzyme activity was assayed by HPLC in the presence of SIL23 or SSI. When M-801 was used as a substrate, formation of M-802 was effectively blocked by any of these inhibitors (Fig. 7). The same activity of SAM-P45 was also inhibited by SIL23 and SSI. Taking this together with the data from native PAGE, it can be concluded that the hydrolyzing activities of DHP-A and SAM-P45 are inhibited by the binding of the proteinaceous inhibitors to the enzymes. This implies that these enzymes use a common catalytic site for both proteolytic and enantioselectively hydrolytic (for 1,4-dihydropyridine diesters) activities. These inhibitor proteins are considered to be a type of nonscissile substrate analogue (6). Study of the tertiary complex of DHP-A with SIL23 would provide a molecular basis for understanding the reaction mechanism of DHP-A toward scissile substrates, such as prochiral 1,4-dihydropyridine diesters M-801 and P-902.



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  		FIG. 7. Inhibition of hydrolysis activity by SSI and SIL23 inhibitors. The HPLC hydrolysis activity assay was carried out by the same procedure described in the legend to Fig. 2. mAbs, milli-absorbance units.


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ACKNOWLEDGMENTS
 
We thank Takuro Tsuruta and Koji Matsuzaki (Bioresource Laboratories in Iwata [Mercian]) for helpful discussions and technical assistance with the HPLC analysis for 1,4-dihydropyridines.


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FOOTNOTES
 
* Corresponding author. Mailing address: Bioresource Laboratories, Mercian Corporation, 4-9-1 Johnan, Fujisawa, Kanagawa 251-0057, Japan. Phone: 81-466-35-1519. Fax: 81-466-35-1524. E-mail: arisawa-a{at}mercian.co.jp. Back

* Corresponding author. Mailing address: School of Agriculture, Meiji University, 1-1-1 Higashi-santa, Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan. Phone: 81-44-934-7831. Fax: 81-44-934-7831. E-mail: staguchi{at}isc.meiji.ac.jp. Back


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Applied and Environmental Microbiology, June 2002, p. 2716-2725, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2716-2725.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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