Probing the Surface of a Laccase for Clues towards the Design of Chemo‐Enzymatic Catalysts

Abstract

Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium–polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues. Distinct ruthenium–polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII–CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.L.S. was the recipient of a MinistHre de l’Education Nationale fellowship.\ud This study was supported by grants from the Agence Nationale de la Recherche (ANR-09-BLANC-0176 and ANR-15-CE07-0021-01) and from the Ministerio de EconomÍa, Industria y\ud Competitividad (CTQ2016-79138-R). We thank Elise Courvoisier-Dezord from the Plateforme AVB (AMU): Analyse et Valorisation\ud de la Biodiversit8 and Yolande Charmasson for help in the production of the recombinant enzymes, as well as Pascal Mansuelle\ud and R8gine Lebrun from the Plateforme Prot8omique (CNRSAMU) for help in acquiring mass spectrometry data.Peer ReviewedPostprint (published version

Photobiocatalysis: Activating Redox Enzymes by Direct or Indirect Transfer of Photoinduced Electrons

“…[109] Tron et al described light-driven, four-electron reduction of dioxygen by laccases. [24,[110][111][112] In the presence of EDTAand [Ru(bpy) 3 ] 2+ ,fully reduced laccase reduced O 2 with aT OF of 18 h À1 . [110] To enhance the photoenzymatic efficiency,azinc porphyrin (ZnTMPyP 4+ )w as used as ap hotosensitizer,b ecause its triplet excited state lifetime is approximately three orders longer than that of [Ru-(bpy) 3 ] 2+ .…”

“…Tron et al. described light‐driven, four‐electron reduction of dioxygen by laccases . In the presence of EDTA and [Ru(bpy) 3 ] 2+ , fully reduced laccase reduced O 2 with a TOF of 18 h −1 .…”

Site directed confinement of laccases in a porous scaffold towards robustness and selectivity

“…C30 is a typical fungal laccase obtained in high yield as a recombinant enzyme. Mono-functionalizable variants of this laccase have been constructed by site-directed mutagenesis [ 43 , 44 ]. Briefly, the only two lysine groups (K 40 and K 71 , Fig.…”

“…Briefly, the only two lysine groups (K 40 and K 71 , Fig. 1 ) present in the original LAC3 polypeptide chain (located opposite of the active T1 center) are first replaced by the most frequent amino acids found at these positions in other laccases (i. e. M 40 , H 71 ) by site directed mutagenesis; the resulting lysine/free variant is then a platform for the substitution of any surface amino acid by a lysine group [ 44 ]. Next, UNIK variants (i.e.…”

Photobiokatalyse: Aktivierung von Redoxenzymen durch direkten oder indirekten Transfer photoinduzierter Elektronen

“…[104] Die Gesamtreaktion der enzymatischen N 2 -Fixierung lautet [Eq. (1)]: [24,[110][111][112] In der Gegenwart von EDTAu nd [Ru(bpy) 3 ] 2+ war vollständig reduzierte Laccase in der Lage, O 2 mit einer TOFv on 18 h À1 zu reduzieren. [110] Um die Effizienz dieser photoenzymatischen Umsetzung zu erhçhen, wurde ein Zinkporphyrin (ZnTMPyP 4+ )a ls Photosensibilisator eingesetzt, dessen angeregter Triplettzustand eine um drei Grçßenordnungen hçhere Lebensdauer besitzt als [Ru-(bpy) 3 ] 2+ .…”