Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

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

General Method for Sequence-independent Site-directed Chimeragenesis

We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined β-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genescreated, and screened the library of 2^8 (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination

Method to protect a targeted amino acid residue during random mutagenesis

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C(30) carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C(40) synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies

Comparison of Library Design to Domains, Dynamics, and Secondary Structure of CYP102A1

<div><p>(A) Crossovers in the library designed using the SCHEMA energy function capture domain boundaries of CYP102A1 determined from molecular dynamics simulations [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040112#pbio-0040112-b027" target="_blank">27</a>]. Crossovers between blocks 2–3, 4–5, 5–6, and 7–8 lie within α-helices. (Secondary structure assignment is based on the CYP102A1 crystal structure [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040112#pbio-0040112-b024" target="_blank">24</a>]). </p> <p>(B) Plot of the RMSD between the backbone atoms of the substrate-bound (closed) and unbound (open) structures of CYP102A1. The RMSD was calculated by comparing molecule B of the substrate-free structure [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040112#pbio-0040112-b029" target="_blank">29</a>] and molecule A of the structure bound to palmitoleic acid [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040112#pbio-0040112-b026" target="_blank">26</a>] using Swiss PDB Viewer. Vertical lines designate crossover locations and blocks are numbered. Crossovers between blocks 1–2, 5–6, 6–7, and 7–8 occur at positions that move < 1.2 Å between the two structures. Crossover 3–4 is located next to a region of high identity and may be shifted towards the N-terminus by up to 14 residues and still produce the same chimeras. This shift allows it to occur at a position which moves < 1.2 Å. </p></div

Structural Model of Heme-Domain Backbone Structure Showing Positions of Each Block

<p>Model is based on the crystal structure of CYP102A1 (2HPD) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040112#pbio-0040112-b026" target="_blank">26</a>]. Blocks are color-coded as shown and heme is shown in CPK coloring. </p