Discovery of a new class of bacterial heme-containing CC cleaving oxygenases.

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t-anethole oxygenase from Stenotrophomonas maltophilia (TAOSm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAOSm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t-anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAOSm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAOSm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry.

Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration.

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD-RIAD) system. These peptides allow self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer-Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD-PTDHRIDD and PAMORIAD/RIAD-PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO-PTDH complex less PTDH was required for the same performance when compared with the PTDH-PAMO fusion enzyme.