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Titolo:
Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant
Autore:
Li, QS; Schwaneberg, U; Fischer, M; Schmitt, J; Pleiss, J; Lutz-Wahl, S; Schmid, RD;
Indirizzi:
Univ Stuttgart, Inst Tech Biochem, D-70569 Stuttgart, Germany Univ Stuttgart Stuttgart Germany D-70569 hem, D-70569 Stuttgart, Germany
Titolo Testata:
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
fascicolo: 1-2, volume: 1545, anno: 2001,
pagine: 114 - 121
SICI:
0167-4838(20010209)1545:1-2<114:REOAMC>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
DIRECTED EVOLUTION; FATTY-ACIDS; P450BM-3; SUBSTRATE; MONOOXYGENASE; ENZYME; DOMAIN; BM3;
Keywords:
cytochrome P-450; rational evolution; mutagenesis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
16
Recensione:
Indirizzi per estratti:
Indirizzo: Schmid, RD Univ Stuttgart, Inst Tech Biochem, Allmandring 31, D-70569 Stuttgart, Germany Univ Stuttgart Allmandring 31 Stuttgart Germany D-70569 ermany
Citazione:
Q.S. Li et al., "Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant", BBA-PROT ST, 1545(1-2), 2001, pp. 114-121

Abstract

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium, was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega -p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega -p-nitrophenoxydecanoic acid (10-pNCA) and omega -p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3. (C) 2001 Elsevier Science B.V. All rights reserved.

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Documento generato il 05/12/20 alle ore 01:48:34