PubMed:11342037 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/11342037","sourcedb":"PubMed","sourceid":"11342037","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/11342037","text":"Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.\nThe 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.","tracks":[{"project":"NCBITAXON","denotations":[{"id":"T1","span":{"begin":130,"end":138},"obj":"OrganismTaxon"}],"attributes":[{"id":"A1","pred":"db_id","subj":"T1","obj":"1386"},{"id":"A2","pred":"db_id","subj":"T1","obj":"55087"},{"subj":"T1","pred":"source","obj":"NCBITAXON"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"NCBITAXON","color":"#93c6ec","default":true}]}]}}