We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

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Cytochrome P450 monooxygenase BM‐3 (EC 1.14.14.1) hydroxylates fatty acids with chain lengths between C12 and C18. It is also known to oxidize the corresponding alcohols and amides. However, it is not known to oxidize alkanes. Here we report that P450 BM‐3 oxidizes octane, which is four carbons shorter and lacks the carboxylate functionality of the shortest fatty acid P450 BM‐3 is known to accept, to 4‐octanol, 3‐octanol, 2‐octanol, 4‐octanone, and 3‐octanone. The rate is much lower than for oxidation of the preferred fatty acid substrates. In an effort to explore the plasticity and mechanisms of substrate recognition in this powerful biocatalyst, we are using directed evolution − random mutagenesis, recombination, and screening − to improve its activity towards saturated hydrocarbons. A spectrophotometric assay has been validated for high throughput screening, and two generations of laboratory evolution have yielded variants displaying up to five times the specific activity of wild‐type P450 BM‐3.

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (A Glieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139; it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidation by 139-3, styrene oxide, reacts with the nucleophile γ-(4-nitrobenzyl)pyridine (NBP) to form a purplecolored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 µM of product, and the detection limit for P450 BM-3 139-3 is~0.2 µM of enzyme. To validate the assay, activities in a small library of random mutants were compared to those determined using an NADPH depletion assay for initial turnover

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

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The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate.