New Reactions And Novel(R)-Selective Enzymes Of Styrene Monooxygenases

Both(S)-and(R)-enantiomers of epoxides are essential intermediates in organic synthesis for active pharmaceuticals,natural products,fine chemicals,and advanced polymeric materials.Chemo-catalyzed methods have excellent stereoselectivity for the epoxidation of internal olefins or olefins with functional groups,but the enantioselectivity for terminal olefins is insufficient.In the biocatalytic arena,styrene monooxygnases(SMO),Cytochrome P450 s,peroxidases,and alkene monooxygenases are reported to catalyze the epoxidation of terminal alkenes with moderate to excellent enantioselectivity.Among them,styrene monooxygnases(SMOs)are known to display almost exclusively epoxygenase activity with outstanding enantioselectivity.However,there are major limitations: almost all known SMOs are(S)-selective,and they mainly catalyze epoxidation and sulfoxidation.Until very recently,our group discovered and reported the first(R)-selective styrene monooxygenases(StStyA)from the genome of Streptomyces NRRL S-31,which can catalyze the epoxidation of styrene and four analogues.The reaction produces the corresponding(R)-epoxide with 91-99%ee.However,its catalytic activity for styrene is low,and the ee value is 91%,while the industrial application usually requires ee value > 98%.At the same time,StStyA can only catalyze meta-substituted styrenes,but it shows no catalytic activity for ortho-and para-substituted styrenes.Therefore,it is necessary to find more novel(R)-selective SMOs.Based on this,this work mainly conducts research from two aspects: the search for novel(R)-selective enzymes and the development of new reactions for SMO.To mine novel(R)-SMOs,we first applied the amino acid sequence of StStyA as a probe,and performed a BLAST search in the NCBI database,which returned a series of functionally unknown protein sequences with a homology of 49-90%.Then phylogenetic analysis was conducted with the obtained sequences together with 28 known(S)-SMOs as well as StStyA.The results showed that the evolutionary tree can be divided into four branches.The typical(S)-SMOs are divided into group I and group II,and the putative SMOs are divided into group III and group IV.A total of 21 new enzymes were selected from group III and group IV as target sequences,and co-expression plasmids encoding each new enzyme and a reductase PsStyB was constructed.The catalytic activity of the new enzymes toward styrene was tested using a whole-cell catalytic system,revealing 10 active new enzymes with 2 of them being(S)-selective and 8 being(R)-selective.All 8 new(R)-SMOs are clustered at Group IV.Gene cluster analysis of those new(R)-SMOs showed that their source strains did not have the styrene degradation gene cluster attached to typical SMOs.Nevertheless,multiple sequence alignment revealed that they have the same conserved domains as typical(S)-SMOs.The alignment also revealed that the enantioselectivity of SMOs appeared in line with the amino acid residue at position 46(AaStyA numbering),which is mainly Ser/Thr and Leu for(S)-SMOs,but Phe for(R)-SMOs.Site-directed mutagenesis studies on both(S)-SMOs and(R)-SMOs showed that this position is indeed critical,and the enantioselectivity of those enzymes could be reversed by mutating position 46 residue.Among the 8 novel(R)-SMOs,three of them,SeStyA from Streptomyces exfoliatus,AaStyA from Amycolatopsis albispora,and PbStyA from Pseudonocardiaceae performed better than others.To further explore their potential,they were carefully characterized.Each enzyme was expressed and purified,yielding 200 mg,60 mg and 15 mg purified enzymes from one liter of culture for SeStyA,AaStyA and PbStyA,respectively.Kinetic analysis of the three enzyms in a reconstituted reaction system showed that their catalytic activities are comparable to known(S)-SMOs.Subsequently,the substrate spectra of SeStyA,AaStyA and PbStyA were tested.Each could catalyze a series of conjugated styrene analogues into corresponding(R)-epoxy products.SeStyA and AaStyA showed higher activity than PbStyA and broader spectrum toward orho-,meta-,para-substituted styrenes,with product enantiomeric excess of up to >99%ee;while PbStyA had extraordinary enantioselectivity,with enantiomeric excess of >99%ee in all cases except one.Interestingly,the enantioselectivity of SeStyA and AaStyA was reversed for para-substituted styrenes,delivering(S)-epoxy products.At the same time,we also investigated the oxidation of other potential substrates using SeStyA as the catalyst,including other styrene analogoues,heterocyclic alkenes,non-conjugated olefins,thioethers,which further expanded the spectrum of(R)-SMOs.To improve the characteristics of(R)-SMOs,SeStyA,with higher activity,was selected for thermal stabilization.The Consensus Finder web tool was used to predict potential stabilizing substitutions,and 12 substitutions with >70% consensus were investigated.Point mutagenesis resulted in 12 single mutants and a double mutant Se M2.After enzymatic assay before and after heat shock,4 single mutants and Se M2 were found to have higher thermal stability than the wild type.After recombination of the 6 mutational positions,the best combinatory mutant,namely Se M6,was obtained.The half-life of inactivation of Se M6 at50?C was 5 times of the wild-type;the activity was around 1.5 times of the wild-type;and the optimal reaction temperature increased from 45?C to 50?C.The expression level of Se M6 was improved too,resulting in 250 mg purified protein per liter of culture.In the epoxidation of4-vinyl-2,3-dihydrobenzofuran,the cell extract of wild-type SeStyA achieved 50% conversion for 10 mmol/L substrate after 6-h incubation at 40 ℃,while Se M6 resulted in complete conversion after 3 h.In addition,Se M6 could achieve 80% conversion for 20 mmol/L substrate after 3-h incubation.It is clearly that the improved mutant would significantly enhance the potential of SeStyA.In the pursuit of new reactions catalyzed with SMO,we were inspired by the mechanism of Achmatowicz oxidative rearrangement,and chose 2-furan-2-propanol as a model substrate to test the activity of several SMOs.Gratifyingly,5 enzymes were able to produce new products.After purification and characterization,the product was proved to be a dihydropyrone,4-hydroxy-6,6-dimethylcyclohex-2-enone,which was resulted from Achmatowicz rearrangement.The reaction was repeated using purified enzymes,which confirmed that SMO can catalyze furanol to produce dihydropyrone.To probe critical amino acid residues involved in Achmatowicz rearrangement,we performed molecular docking of the substrate into PsStyA,and constructed point mutants based on their interaction with the substrate.Enzymatic assay showed that the majority of the positions tested had great influence on Achmatowicz rearrangement.In summary,this study has revealed for the first time a new cluster of(R)-SMOs,and several novel(R)-SMOs have been characterized,providing a valuable green alternative to the synthetic tool box for enantiopure(R)-epoxides.The discovery of the SMO-catalyzed Achmatowicz rearrangement is a breakthrough from commonly known reactions,which would tremendously expand the application of SMO.