Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450
Abstract
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 possible structural differences between parents CYP102A1 and CYP102A2.
Additional Information
© 2006 Otey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The NCBI Entrez (http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi) accession numbers for the genes and gene products discussed in this paper are CYP102A1 (J04832), CYP102A2 (CAB12544) and CYP102A3 (U93874). We thank Kiowa S. Bower for laboratory assistance. CRO, ML, KH, and FHA conceived and designed the experiments. CRO, ML, and KH performed the experiments. CRO, ML, JBE, JDB, and FHA analyzed the data. JBE, JDB, and FHA contributed reagents/materials/analysis tools. CRO, ML, and FHA wrote the paper. The authors have declared that no competing interests exist.Attached Files
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Additional details
- PMCID
- PMC1431580
- Eprint ID
- 2699
- Resolver ID
- CaltechAUTHORS:OTEpbio06
- Created
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2006-04-20Created from EPrint's datestamp field
- Updated
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2021-11-08Created from EPrint's last_modified field