Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: Production of an efficient enzyme for bioconversion of fine chemicals

dc.contributor.author	Axarli, I	en
dc.contributor.author	Prigipaki, A	en
dc.contributor.author	Labrou, NE	en
dc.date.accessioned	2014-06-06T06:46:42Z
dc.date.available	2014-06-06T06:46:42Z
dc.date.issued	2005	en
dc.identifier.issn	13890344	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.bioeng.2004.11.003	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/3148
dc.subject	Biotransformation	en
dc.subject	BM3	en
dc.subject	CYP	en
dc.subject	Directed evolution	en
dc.subject	Enzyme immobilization	en
dc.subject	P450	en
dc.subject	substrate recognition	en
dc.subject.other	Bioassay	en
dc.subject.other	Biochemical engineering	en
dc.subject.other	Bioconversion	en
dc.subject.other	Catalysis	en
dc.subject.other	Cell immobilization	en
dc.subject.other	Cells	en
dc.subject.other	Enzymes	en
dc.subject.other	Escherichia coli	en
dc.subject.other	Fatty acids	en
dc.subject.other	Mutagenesis	en
dc.subject.other	Polypeptides	en
dc.subject.other	Synthesis (chemical)	en
dc.subject.other	Activity assays	en
dc.subject.other	Aromatic substrates	en
dc.subject.other	Cytochromes	en
dc.subject.other	Cytoplasmic enzymes	en
dc.subject.other	Cytology	en
dc.subject.other	1,4 naphthoquinone derivative	en
dc.subject.other	aminocaproic acid	en
dc.subject.other	cytochrome P450	en
dc.subject.other	cytochrome p450 cyp102a2	en
dc.subject.other	dodecyl sulfate sodium	en
dc.subject.other	epoxide	en
dc.subject.other	etacrynic acid	en
dc.subject.other	lauric acid	en
dc.subject.other	mutant protein	en
dc.subject.other	proline	en
dc.subject.other	sepharose	en
dc.subject.other	serine	en
dc.subject.other	unclassified drug	en
dc.subject.other	unspecific monooxygenase	en
dc.subject.other	alkalinity	en
dc.subject.other	amino terminal sequence	en
dc.subject.other	article	en
dc.subject.other	beta sheet	en
dc.subject.other	binding site	en
dc.subject.other	bioreactor	en
dc.subject.other	biotransformation	en
dc.subject.other	controlled study	en
dc.subject.other	directed molecular evolution	en
dc.subject.other	enzyme activity	en
dc.subject.other	enzyme assay	en
dc.subject.other	enzyme binding	en
dc.subject.other	enzyme engineering	en
dc.subject.other	enzyme mechanism	en
dc.subject.other	enzyme specificity	en
dc.subject.other	enzyme stability	en
dc.subject.other	Escherichia coli	en
dc.subject.other	low temperature procedures	en
dc.subject.other	molecular model	en
dc.subject.other	nonhuman	en
dc.subject.other	nucleotide sequence	en
dc.subject.other	oxidation	en
dc.subject.other	polymerase chain reaction	en
dc.subject.other	prediction	en
dc.subject.other	priority journal	en
dc.subject.other	protein domain	en
dc.subject.other	protein expression	en
dc.subject.other	protein immobilization	en
dc.subject.other	protein interaction	en
dc.subject.other	Amino Acid Sequence	en
dc.subject.other	Bacillus subtilis	en
dc.subject.other	Bacterial Proteins	en
dc.subject.other	Biotransformation	en
dc.subject.other	Cytochrome P-450 Enzyme System	en
dc.subject.other	Directed Molecular Evolution	en
dc.subject.other	Fatty Acids	en
dc.subject.other	Molecular Sequence Data	en
dc.subject.other	Oxidation-Reduction	en
dc.subject.other	Polycyclic Hydrocarbons, Aromatic	en
dc.subject.other	Substrate Specificity	en
dc.title	Engineering the substrate specificity of cytochrome P450 CYP102A2 by directed evolution: Production of an efficient enzyme for bioconversion of fine chemicals	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.bioeng.2004.11.003	en
heal.publicationDate	2005	en
heal.abstract	The P450 cytochromes constitute a large family of hemoproteins that catalyze the monooxygenation of a diversity of hydrophobic substrates. CYP102A2 is a catalytically self-sufficient cytoplasmic enzyme from Bacillus subtilis, containing both a monooxygenase domain and a reductase domain on a single polypeptide chain. CYP102A2 was subjected to error-prone PCR to generate mutants with enhanced activity with fatty acids and other aromatic substrates. The library of CYP102A2 mutants was expressed in BL21(DE3) Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. After a single round of error-prone PCR, the variant Pro15Ser exhibiting modified substrate specificity was generated. This variant showed approximately 6- to 9-fold increased activity with SDS, lauric acid and 1,4-naphthoquinone, and enhanced activity for other substrates such as ethacrynic acid and ε-amino-n-caproic acid. Molecular modeling of the CYP102A2 monooxygenase domain suggested that Pro15 is located in a short helical segment and is involved in extensive interactions between the N-terminal domain and the β2 sheet, which contribute to the formation of the substrate binding site. Thus, Pro15 appears to affect substrate binding and catalysis indirectly. These results clearly demonstrate the importance of remote residues, not readily predicted by rational design, for the determination of substrate specificity. In addition, we report here that the Pro15Ser variant of CYP102A2 can be efficiently immobilized on epoxy-activated Sepharose at pH 8.5 and 4°C. The immobilized variant of CYP102A2 retains most of its activity (81%) and shows improved stability at 37°C. The approach offers the possibility of designing a P450 bioreactor that can be operated over a long period of time with high efficiency and which can be used in fine chemical synthesis. © 2005 Elsevier B.V. All rights reserved.	en
heal.journalName	Biomolecular Engineering	en
dc.identifier.issue	1-3	en
dc.identifier.volume	22	en
dc.identifier.doi	10.1016/j.bioeng.2004.11.003	en
dc.identifier.spage	81	en
dc.identifier.epage	88	en