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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 (TAO Sm). 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 TAO Sm 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 TAO Sm 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, TAO Sm 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. • Identification of a new class of heme-containing enzymes. • Cofactor-independent biocatalytic C C cleavage. • Crystal structure of a trans-anethole oxygenase. • Novel active site architecture of a heme-containing enzyme. [ABSTRACT FROM AUTHOR]

Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study on six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted, and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acid side chains coordinate to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases. [ABSTRACT FROM AUTHOR]