Artificial Family of Cytochrome p450Edit
The understanding of how a protein operates both structurally and functionally is done by the comparison of many sequences that are related. The creation of artificial proteins gives a more indepth understanding of sequence and functional diversity. These artificial proteins allow us to understand functions that may not be important biologically, but will give a more broad understanding of the proteins themselves. To do this, the researchers used a program known as SCHEMA that creates libraries of sequences that are highly mutated and have a likelihood of folding into a structure similar to the parental structure. In this particular creation, three cytochrome p450 amino acid sequences went through SHEMA- guided recombination to create roughly ~3000 properly folding proteins or chimeras (Otey et al. 2006).The artificial family was created by taking sections of the cytochrome p450 heme- binding domain. The three proteins used were bacterial cytochrome p450's 102A1, 102A2, and 102A3.
Chimera testing in comparison to parent proteinsEdit
After the creation of these proteins, the catalytic activities of human engineered cytochrome p450 enzymes were analyzed by reacting 320 of these chimeras with 2- phenoxyethanol to see if there was peroxygenase activity like in the parent proteins. These monooxygenases used NADPH and molecular oxygen to hydroxylate fatty acids to phenol. Of the 320 proteins created, 72% were found to be active on the 2- phenoxyethanol.
The thermostability of 14 chimeras were also tested to see if the recombination affected protein stability, all of which denatured at a high temperature. The range of protein denaturation was found to be between 42 and 62 degrees celsius. The most stable chimera was found to have 84 amino acid substitution differences from the closest parent, but the denaturing temperature was only 7 degrees higher than the most stable parent.Overall, This protein family developed in the laboratory is helpful to identify regions of the amino acid sequence that are important to folding in function. Clearly, in the cytochrome p450 protein, the heme- binding domain is essential to the monooxygenase reaction that the cytochrome p450 protein often catalyzes.
- Alessandra: Cytochrome p450: Introduction
- Alessandra: Cytochrome p450: Biological function
- Alessandra: Cytochrome p450: Biosynthesis
- Alessandra: Cytochrome p450: Gene sequence
- Alessandra: Cytochrome p450: Amino acid sequence and composition
- Alessandra: Cytochrome p450: Secondary and tertiary structure
- Alessandra: Cytochrome p450: Domains and structural motifs
- Alessandra: Cytochrome p450: Interactions with macromolecules and small molecules
- Alessandra: Cytochrome p450: Molecular biodiversity and evolution
- Alessandra: Cytochrome p450: Literature overview
- Alessandra: Cytochrome p450: Useful online resources
Otey, Christopher R., Marco Landwehr, Jeffrey B. Endelman, Kaori Hiraga, Jesse D. Bloom, and Frances H. Arnold. “Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450.” National Center for Biotechnology Information. U.S. National Library of Medicine, 11 Apr. 2006. Web. 12 Feb. 2014.