Streptomyces serine protease (DHP-A) as a new biocatalyst capable of forming chiral intermediates of 1,4-dihydropyridine calcium antagonists.
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.
Arisawa
Akira
A
Bioresource Laboratories, Mercian Corporation, Fujisawa, Kanagawa 251-0057, Japan. arisawa-a@mercian.co.jp
Matsufuji
Motoko
M
Nakashima
Takashi
T
Dobashi
Kazuyuki
K
Isshiki
Kunio
K
Yoshioka
Takeo
T
Yamada
Shigeru
S
Momose
Haruo
H
Taguchi
Seiichi
S
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004095:Dihydropyridines / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D004095:Dihydropyridines; C038806:1,4-dihydropyridine; D012697:Serine Endopeptidases