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Title: Mutagenesis studies to assess substrate specificity determinants of multicopper oxidases

Speaker: Vânia Brissos

Affiliation: Microbial & Enzyme Technology, ITQB NOVA

Abstracts: Laccases are green catalysts with an outstanding redox capability over a wide range of aromatic substrates, using O2 as an electron acceptor and releasing water as reduced product [1]. We have recently reported a laboratory evolution approach that led to the improved efficiency of a hyperthermophile bacterial metallo-oxidase for aromatic compounds [2]. The 2B3 variant of McoA from Aquifex aeolicus, showing 10 non-synonymous mutations, featured a 2-order of magnitude higher efficiency than the wild-type for the typical laccase substrate ABTS (2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)) while showing an enhanced solubility and thus a higher kinetic and thermodynamic thermostability. In order to investigate the structural determinants of substrate specificity underlying laccase and metallo-oxidase function, mutagenesis studies, X-ray crystal and Small-angle X-ray scattering (SAXS) were performed. First the random recombination of the 2B3 and wild-type McoA genes was performed to discriminate neutral from functional mutations; 2F4 hit variant was selected with 6 non-synonymous beneficial mutations (M449T, I441L, R471G, I199T, P58S and F55S). X-ray crystal studies indicate that both 2F4 and wild-type enzymes share the typical overall fold of multicopper oxidases; the structures show a similar spatial arrangement of the T1-Cu center and the trinuclear cluster. A long Met rich loop over the T1- Cu center, known as Met-rich region [3], is not visible in the structures of both enzymes probably due to its higher mobility; however, small structural rearrangements near M449T are observed in the 2F4 variant, indicating a different conformation of this loop relatively to the wild-type McoA. SAXS studies show that the Met rich loop is occluding the T1-Cu center, turning the entrance to the active site statistically less accessible in the wild-type, whereas in the 2F4 variant, a structural rearrangement resulted in a more open conformation that putatively facilitates the entrance of aromatic compounds. In an attempt to discriminate the role of the loop in the substrate’s catalytic efficiency we have deleted the loop from both wild-type and 2F4 variant. These variants were biochemically characterized and structurally validated by SAXS. The effect of each mutation of the 2F4 variant will be discussed based on the biochemical analysis of multiple variants constructed by site-directed mutagenesis (evolution pathway reconstruction).

[1] Martins L.O.; Durão P.; Brissos V.; Lindley P.F. Cell Mol Life Sci 2015, 77, 911-922. [2] Brissos V.; Ferreira M.; Grass G.; Martins L.O. ACS Catalysis 2015, 5, 4932-4941. [3] Roberts S.A.; Weichsel A.; Grass G.; Thakali K.; Hazzard J.T.; Tollin G.; Rensing C.; Montfort W.R. Proc Natl Acad Sci USA 2002, 99, 2766-2771.