Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use

Abstract

The invention relates to novel variants of cytochrome P450 oxygenases. These variants have at least one mutation improving their ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. The variants also have at least one mutation improving thermostability as compared to the parent enzyme or corresponding wild-type enzyme. Preferred variants include cytochrome P450 BM-3 heme domain variants having L52I, I58V, F87A, H100R, S106R, F107L, A135S, M145A / V, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and / or V446I amino acid substitutions.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]This invention relates to variants of cytochrome P450 oxygenases. Specifically, the invention relates to thermostable variants of cytochrome P450 oxygenases capable of improved peroxide-driven hydroxylation, and methods of making thermostable variants.[0003]2. Background Information[0004]One of the great challenges of contemporary catalysis is the controlled oxidation of hydrocarbons. Processes for controlled, stereo- and regioselective oxidation of hydrocarbon feed stocks to more valuable and useful products such as alcohols, ketones, acids, and peroxides would have a major impact on the chemical and pharmaceutical industries. However, selective oxyfunctionalization of hydrocarbons remains one of the great challenges for contemporary chemistry. Despite decades of effort, including recent advances, the insertion of oxygen into unactivated carbon-hydrogen bonds (hydroxylation) remains difficult to achieve with high selec...

AI Technical Summary

Benefits of technology

The present invention is based on the discovery of mutations in a protein called P450 BM-3 that improve its ability to use peroxide as an oxygen source. These mutations also increase the thermostability of the protein. The invention provides a method for thermostabilizing a variant of P450 BM-3 by adding a second mutation in a nearby amino acid residue. The resulting variant has a higher ability to utilize peroxide and is more stable than the wild-type enzyme. The invention also provides a protein library of variants of P450 BM-3 with the second mutation that exhibit higher thermostability than the wild-type enzyme. Overall, the invention provides a way to improve the function and stability of P450 BM-3 for use in industrial applications.

Problems solved by technology

One of the great challenges of contemporary catalysis is the controlled oxidation of hydrocarbons.

However, selective oxyfunctionalization of hydrocarbons remains one of the great challenges for contemporary chemistry.

Despite decades of effort, including recent advances, the insertion of oxygen into unactivated carbon-hydrogen bonds (hydroxylation) remains difficult to achieve with high selectivity and high yield.

Many chemical methods for hydroxylation require severe conditions of temperature or pressure, and the reactions are prone to over-oxidation, producing a range of products, many of which are not desired.

Most impressive is their ability to catalyze the specific hydroxylation of non-activated C—H, one of the most useful biotransformation reactions, which is often difficult to achieve by chemical means, especially in water, at room temperature and atmospheric pressure.

Unfortunately, P450s are generally limited by low turnover rates, and they generally require an expensive cofactor, NADH or NADPH, and at least one electron transfer partner protein (reductase).

Furthermore, the enzymes are large, complex, and expensive.

Wild-type P450s are in some cases capable of using peroxides as a source of oxygen and electrons via a peroxide “shunt” pathway, though the efficiency of this route is low.

However, low efficiency is a major limitation.

Further, wild-type enzymes capable of peroxide-driven hydroxylation, such as chloroperoxidase (CPO) and CYP152B1 are generally limited in their substrate specificity to hydroxylation of activated C—H bond carbons, i.e., carbon atoms adjacent to a functional group such as an aromatic ring, a carbonyl group, a heteroatom, etc.

While the activity of enzymes has thus been improved and modified, a continuing problem is that enzymes are often poorly stable under conditions encountered during production, storage or use.

Stabilizing the relatively unstable cytochrome P450 enzymes by protein engineering is a particularly challenging problem, however, partly because the P450s comprise multiple subunits and contain thermolabile co-factors.

Unfortunately, the functions of these P450s are not known, and reported activities are low (e.g., 0.35 min−1 in the NADH-driven hydroxylation of lauric acid.

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