Science 339, 307–310 (2013)

Synthetic chemists have developed a diverse range of metal-catalysed cyclopropanation reactions that involve the addition of a carbene to an olefin. Despite the fact that iron–porphyrin centres, which are common in metalloenzymes, will catalyse this reaction under the right conditions, there are no enzymes that harness similar chemistry for cyclopropanation in their natural biological environment. Some enzymes, however, are known to be promiscuous enough to react with alternative reactants to catalyse a different chemical reaction under the right conditions — albeit normally at a much lower level of activity.

In an effort to see if any metalloenzymes were promiscuous enough to adapt to metallocarbene chemistry, a team led by Frances Arnold at the California Institute of Technology screened a selection of naturally occurring haem proteins (which contain an iron–porphryin moiety) to see if they could be induced to catalyse a cyclopropanation reaction between styrene and ethyl diazoacetate — common reactants for testing catalysts for this transformation. It was found that several of the enzymes could catalyse the cyclopropanation, albeit at low levels. Of these enzymes, cytochrome P450BM3 (a monooxygenase from the soil bacterium Bacillus megaterium) produced a mixture of cis and trans diasteroisomers and seemed to be the best candidate for further optimization.

The next step was to identify mutations that improved the catalytic properties. Screening a library of 92 variants resulted in five promising candidates that were further analysed to discover which mutations improved the catalytic turnover as well as the diastereo- and enantioselectivity. Based on these results, Arnold and co-workers started to design new variants that combined advantageous mutations. Designing small-molecule catalysts that favour the cis product rather than the trans isomer is more challenging and so the team elected to see if a P450BM3-based metalloenzyme could be adapted to tackle this problem. Mutations to the amino acids around the active site produced enzymes with enhanced cis selectivity and further experiments showed that the variant P450BM3 enzymes could tolerate a range of substrates and functional groups.