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

Diversification of Catalytic Function in a Synthetic Family of Chimeric Cytochrome P450s

We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a “surrogate” for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping

Enantioselective α-Hydroxylation of 2-Arylacetic Acid Derivatives and Buspirone Catalyzed by Engineered Cytochrome P450 BM-3

Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the α-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals. Introduction Enzymes are Nature's catalysts, tremendously accelerating the rates of a wide range of biochemical reactions, often with exquisite specificity. Harnessing enzymes for other purposes usually requires engineering them to improve their activity or stability. One approach to engineering enzymes is to make specific modifications, but this demands a detailed and frequently unattainable understanding of the relationship between sequence and function. Directed evolution bypasses this problem in much the same way as natural evolution, by combining mutation with selection or screening to identify improved variants. Because it is never possible to test more than an infinitesimal fraction of the vast number of possible protein sequences, it is essential to have a strategy for creating directed evolution sequence libraries that are rich in proteins with the desired enzymatic function. Such libraries can be designed by drawing on our knowledge of how proteins respond to mutation Directed evolution strategies Directed evolution works when the researcher can find at least one enzyme with improved properties in the sequence library. The most naïve strategy of creating a library of random protein sequences is not useful for most enzyme engineering goals. Although sequences with simple functions such as ATP binding Most directed evolution strategies involve making relatively small changes to existing enzymes. This takes advantage of the fact that enzymes often have a range of weak promiscuous activities that are quickly improved with just a few mutations Random mutagenesis The most straightforward strategy for library construction is to randomly mutate the full gene of an enzyme with a function close to the desired function. This approach requires no structural or mechanistic information, and can uncover unexpected beneficial mutations. Using sequential rounds of error-prone PCR to make an average of a few mutations per gene, followed by screening or selection for improved variants, is effective for a wide range of engineering goals. The creation of enantioselective catalysts from an enzyme whose structure is unknown is one such application. A single round of error-prone PCR produced several dozen cyclohexane monooxygenases with R or S selectivity Beneficial mutations found by random mutagenesis can be combined by DNA shuffling. A study with b-glucuronidase showed that beneficial mutations drive each other to extinction during recursive random mutagenesis, but that this problem can be eliminated by DNA shuffling Random mutagenesis can also uncover additional beneficial mutations in rationally designed enzymes. The Withers laboratory Targeted mutagenesis Some engineering goals, such as dramatically altering an enzyme's specificity or regioselectivity, may require mul- Random mutagenesis, targeted mutagenesis and recombination are three strategies for producing sequence libraries for directed evolution. (a) Random mutagenesis introduces amino acid substitutions throughout the protein and can uncover beneficial mutations distant from the active site. The red residues in the structure at top show four mutations uncovered by random mutagenesis that enhanced the activity of mammalian cytochrome P450 2B1 on several substrates Using a high-resolution crystal structure to target mutagenesis to three active site residues, Hill et al. [23] created a triple mutant of phosphotriesterase with a rate enhancement of three orders of magnitude for the degradation of organic triesters such as those used in chemical warfare agents. Crucially, two of the corresponding single mutants did not increase activity and so would not have been identified if they had been explored one at a time. The problem of inverting the enantioselectivity of a lipase offers an interesting comparison between full-gene random mutagenesis and targeted mutagenesis. Reetz and co-workers [24] used several rounds of full-gene random mutagenesis and DNA shuffling to invert the enantioselectivity of a lipase of unknown structure from S to R. Another lipase was engineered for the same goal by simultaneous mutation of four active site residues A variety of other enzymes have recently been engineered by targeted mutagenesis. Mutating three active site residues of penicillin acylase created six variants with improved activity, five of which were triple mutants [27]. Juillerat et al. [28] targeted four active site residues to engineer an O6-alkylguanine-DNA alkyltransferase for the efficient in vivo labeling of fusion proteins. They developed a selection system that allowed them to examine over 20,000 mutants and found that the best variants were triple mutants, suggesting the importance of simultaneously exploring multiple mutations. Novel DNA and RNA polymerases have also been engineered by targeted mutagenesis. Chelliserrykattil and Ellington [29] mutated four amino acids in RNA polymerase to engineer the enzyme to transcribe 2 0 -O-methyl RNA. Using a screen that selected variants that generated more RNA, they identified several mutants that incorporated nucleotides modified at the 2 0 position. Fa et al. [30] used targeted mutagenesis to engineer a DNA polymerase to specifically incorporate 2 0 -O-methyl ribonucleoside triphosphates by mutating six amino acids and selecting improved variants using phage display. Targeted mutagenesis of two active site residues was used to engineer a thioredoxin protein to replace the disulfide bond formation system in Escherichia coli Schultz and co-workers have created tRNA synthetases that charge orthogonal tRNAs with non-natural amino acids by targeting mutagenesis to five or six amino acids involved in substrate recognition. They then performed a positive selection for recognition of the non-natural amino acid and a negative selection against recognition of other amino acids The best mutants discovered by targeted mutagenesis almost always contain multiple mutations. These mutations are often beneficial as single mutants, but evidence is accumulating that at least some of them are beneficial only in combination Recombination Recombining structurally similar proteins can access larger degrees of sequence change than random mutagenesis The family shuffling protocol relies on regions of sequence identity to create crossovers that recombine the sequences of related proteins. This protocol is therefore limited to proteins with more than 70-75% identity, because libraries created from more diverged sequences tend to yield mostly parent sequences. A variety of methods have been developed to avoid this problem in the recombination of divergent sequences by using mismatched PCR primer pairs Although the studies described above demonstrate that recombining highly diverged but homologous sequences can produce libraries of diverse folded sequences, so far there has been little work to test whether it is also a useful method for discovering new functions. A tantalizing hint is that four out of fourteen chimeras of two cytochrome P450 proteins with 64% sequence identity show new product profiles Non-homologous recombination that combines fragments of unrelated proteins is another way to introduce large sequence changes. A new methodology was used to recombine the non-homologous chorismate mutase and fumarase proteins A striking application of non-homologous recombination is Ostermeier and co-workers' creation of a protein that combines the activity of a b-lactamase with the maltoseinduced conformational change of maltose-binding protein. In one experiment, they randomly inserted the lactamase sequence into the maltose-binding protein and screened for mutants with enhanced lactamase activity in the presence of maltose Conclusions Directed evolution is now an established method to engineer enzymes for a wide range of uses. Full-gene random mutagenesis continues to be a straightforward and powerful tool, and studies using this approach repeatedly illustrate that beneficial mutations can occur at unexpected sites. Targeted mutagenesis and recombination can extend directed evolution to the engineering of enzyme properties that require more than a few uncoupled changes in a protein's sequence (which are easily obtained by sequential rounds of random mutagenesis and screening). The increasing incorporation of structural and chemical knowledge will undoubtedly enhance the utility of these methods. The growing use of rational design in conjunction with directed evolution offers the exciting promise of generating libraries containing a high frequency of sequences with the desired functional properties. Update Recent work has emphasized the tendency of directed evolution to improve weak promiscuous functions by broadening specificity, as discussed i

Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine

The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h's paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.Charles A. Dana Foundation. Brain and Immuno-ImagingRaymond and Beverley Sackler FoundationNational Institutes of Health (U.S.) (grant R01-DA28299)National Institutes of Health (U.S.) (grant DP2-OD2441)National Institutes of Health (U.S.) (grant R01-GM068664)Jacobs Institute for Molecular Engineering for Medicine. Jacobs Institute for Molecular Engineering for MedicineNational Institutes of Health (U.S.) (grant R01-DE013023

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Preparation of human metabolites of propranolol using laboratory‐evolved bacterial cytochromes P450

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the “peroxide shunt” pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost‐effective, and scaleable route to the synthesis of drug metabolites

Preparation of human metabolites of propranolol using laboratory‐evolved bacterial cytochromes P450

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the “peroxide shunt” pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost‐effective, and scaleable route to the synthesis of drug metabolites

Functional Evolution and Structural Conservation in Chimeric Cytochromes P450: Calibrating a Structure-Guided Approach

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome P450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded P450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse P450s for exploring function evolution within the P450 structural framework