structure-guided recombination creates an artificial family of cytochromes p450structure-guided重组创造了一个人工细胞色素p450的家庭.pdfVIP

PLoS BIOLOGY Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450 1 2 3 2¤ 2 1,2,3* Christopher R. Otey , Marco Landwehr , Jeffrey B. Endelman , Kaori Hiraga , Jesse D. Bloom , Frances H. Arnold 1 Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, United States of America, 2 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America, 3 Bioengineering, California Institute of Technology, Pasadena, California, United States of America Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ;3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (.73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence- structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests