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Titolo:
PURIFICATION AND CLONING OF A PROLINE 3-HYDROXYLASE, A NOVEL ENZYME WHICH HYDROXYLATES FREE L-PROLINE TO CIS-3-HYDROXY-L-PROLINE
Autore:
MORI H; SHIBASAKI T; YANO K; OZAKI A;
Indirizzi:
KYOWA HAKKO KOGYO CO LTD,TOKYO RES LABS,3-6-6 ASAHIMACHI MACHIDA TOKYO 194 JAPAN KYOWA HAKKO KOGYO CO LTD,TOKYO RES LABS MACHIDA TOKYO 194 JAPAN
Titolo Testata:
Journal of bacteriology
fascicolo: 18, volume: 179, anno: 1997,
pagine: 5677 - 5683
SICI:
0021-9193(1997)179:18<5677:PACOAP>2.0.ZU;2-N
Fonte:
ISI
Lingua:
ENG
Soggetto:
STREPTOMYCES; PROTEINS; 4-HYDROXYLASE; BIOSYNTHESIS; QUANTITIES; MECHANISM; SEQUENCE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
22
Recensione:
Indirizzi per estratti:
Citazione:
H. Mori et al., "PURIFICATION AND CLONING OF A PROLINE 3-HYDROXYLASE, A NOVEL ENZYME WHICH HYDROXYLATES FREE L-PROLINE TO CIS-3-HYDROXY-L-PROLINE", Journal of bacteriology, 179(18), 1997, pp. 5677-5683

Abstract

Proline 3-hydroxylase was purified from Streptomyces sp, strain TH1, and its structural gene was cloned, The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H, Mori, T, Shibasaki, Y, Uosaki, K. Ochiai, and A, Ozaki, Appl, Environ, Microbiol. 62:1903-1907, 1996), The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively, The K-m values were 0.56 and O.11 mM for L-proline and 2-oxoglutarate, respectively, The k(cat) value of hydroxylation was 3.2 s(-1). Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database, A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence, With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR, A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe, The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame(ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158, No sequence homolog was found in EMBL, GenBank, and DDBJ databases, ORF 3 was expressed in Escherichia coli DH1, Recombinants showed hydroxylating activity five times higher than that ofthe original bacterium, Streptomyces sp, strain TH1, It was concludedthat the ORF 3 encodes functional proline 3-hydroxylase.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/04/20 alle ore 00:06:22