High-Throughput Screen for Aromatic Hydroxylation

Otey, Christopher R.; Joern, John M.

doi:10.1385/1-59259-396-8:141

Cited by 16 publications

(19 citation statements)

References 15 publications

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“…Reaction on this substrate yields phenol ( Figure 5), which is detectable in high throughput [ 35]. The three parent P450s naturally occur as fusion proteins to an FAD- and FMN-containing NADP reductase [ 21].…”

Section: Resultsmentioning

confidence: 99%

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Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

et al. 2006

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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.

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Section: Resultsmentioning

confidence: 99%

“…Activity on 2-phenoxyethanol was assayed in 96-well plates using the 4-aminoantipyrine assay (4-AAP), which detects phenol-like compounds [ 35]. Reactions were carried out in 120 μl with 1% DMSO, 1% acetone, 100 mM 2-phenoxyethanol and 20 mM H 2 O 2 in 100 mM N-[2-hydroxyethyl]piperazine-N′-[3-propanesulfonic acid] (EPPS) [pH 8.2].…”

Section: Methodsmentioning

confidence: 99%

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

et al. 2006

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“…Hydroxylation of each substrate, at the appropriate positions, leads to phenolic by-products. These phenolic compounds can be coupled to 4-aminoantipyrene (4-AAP) to form a red compound, which is detectable at 500 nm (41). The "enzyme activity" values are the raw absorbance increase at 500 nm, which is proportional to the total substrate turnovers per enzyme after 2 h. All measurements were performed in triplicate and the median values are reported.…”

Section: Methodsmentioning

confidence: 99%

Navigating the protein fitness landscape with Gaussian processes

Romero

Krause

Arnold

2012

Proc. Natl. Acad. Sci. U.S.A.

302

311

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Knowing how protein sequence maps to function (the "fitness landscape") is critical for understanding protein evolution as well as for engineering proteins with new and useful properties. We demonstrate that the protein fitness landscape can be inferred from experimental data, using Gaussian processes, a Bayesian learning technique. Gaussian process landscapes can model various protein sequence properties, including functional status, thermostability, enzyme activity, and ligand binding affinity. Trained on experimental data, these models achieve unrivaled quantitative accuracy. Furthermore, the explicit representation of model uncertainty allows for efficient searches through the vast space of possible sequences. We develop and test two protein sequence design algorithms motivated by Bayesian decision theory. The first one identifies small sets of sequences that are informative about the landscape; the second one identifies optimized sequences by iteratively improving the Gaussian process model in regions of the landscape that are predicted to be optimized. We demonstrate the ability of Gaussian processes to guide the search through protein sequence space by designing, constructing, and testing chimeric cytochrome P450s. These algorithms allowed us to engineer active P450 enzymes that are more thermostable than any previously made by chimeragenesis, rational design, or directed evolution.protein engineering | recombination | machine learning | experimental design | active learning I n the mapping of protein sequence to protein behavior, the phenotype can be envisioned as a surface, or landscape, over the high-dimensional space of possible sequences (1). This "fitness landscape" could describe how the protein contributes to organismal fitness, or it may represent a biophysical property, such as stability, enzyme activity, or ligand binding affinity. The structure of this surface describes the spectrum of possible phenotypes as well as the mutational accessibility among them and therefore strongly influences protein evolution. This surface is also the objective function for protein engineering, which seeks to identify protein sequences that are highly optimized for a given property or set of properties.Identifying such optimized sequences is extremely challenging for several reasons. First, the space of possible protein sequences is incomprehensibly large and will never be searched exhaustively by any means, naturally, in the laboratory, or computationally (2, 3). Second, within this vast space, functional proteins are extremely scarce, with estimates that range from a high of 1 in 10 11 to as little as 1 in 10 77 (4,5). Of the sequences that are functional, most have poor fitness and their numbers decrease exponentially with higher levels of fitness (6, 7). Thus, highly fit sequences are vanishingly rare and overwhelmed by nonfunctional and mediocre sequences.Computational protein engineering uses models of protein function to guide a search for optimized sequences. These models typically contain an atomic struc...

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“…The activities on 2PE, PROP, 11PA, 2A5C, and MDOB were determined using the 4-aminoantipyrene (4-AAP) assay [59,60], which detects the formation of phenolic compounds. For each of these five substrates, immediately before use we prepared a 6× substrate stock solution.…”

Section: Methodsmentioning

confidence: 99%

Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

et al. 2007

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Background: Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's original biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution.

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High-Throughput Screen for Aromatic Hydroxylation

Cited by 16 publications

References 15 publications

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Navigating the protein fitness landscape with Gaussian processes

Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

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