Substrate and Dioxygen Binding to the Endospore Coat Laccase from Bacillus subtilis

Abstract

The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the noncatalytic co-oxidant 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) belong to the multi-copper oxidase family of enzymes that includes ascorbate oxidase (L-ascorbate oxygen oxidoreductase; EC 1.10.3.3) and ceruloplasmin (Fe(II) oxygen oxidoreductase; EC 1.16.3.1); for recent reviews, see Refs. 1 and 2). X-ray structural studies over the past decade have enabled the elucidation of a significant number of structural and functional aspects of these enzymes, but many key questions still remain. Two of these involve the mechanism of electron transfer from substrates and the mechanism of dioxygen reduction (2, 3). Laccases are the simplest representatives of the family and are therefore used as model systems for investigating structurefunction relationships in the multi-copper oxidases. Such fundamental studies are aimed at clarifying the molecular basis for the enzyme redox potential and the specificity toward different substrates.Most of the known laccases have fungal (e.g. white-rot fungi) or plant origins, although a few laccases have recently been identified and isolated in bacteria (4 -6). These enzymes have been implicated in many diverse physiological functions such as morphogenesis, pathogenesis, lignin synthesis, and lignolysis (4, 7). Chemically, all these functions are related to the oxidation of a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, diamines, and even some inorganic compounds. The one-electron oxidation of these reducing substrates occurs concomitantly with a four-electron reduction of molecular dioxygen to water. The catalytic centers consist of three structurally and functionally distinct copper centers. T1 copper ("blue copper") is a mononuclear center involved in substrate oxidation, whereas T2 and T3 form a trinuclear center involved in dioxygen reduction to water; for a definition of the copper types, see Ref. 8. The initial substrate reaction products are dioxygen-centered radicals or cation radicals, which usually react further through non-enzymatic routes for the oxidative coupling of monomers or the degradation of polymers.Because of their high relative nonspecific oxidation capacity, laccases have been found to be useful biocatalysts for diverse biotechnological applications (9). Their biotechnological importance showed a marked increased after the discovery that the oxidizing reaction substrate range could be further extended in the presence of the so-called mediators, small readily oxidizable molecules (10)...

Structural Insights into 2,2′-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)-Mediated Degradation of Reactive Blue 21 by Engineered Cyathus bulleri Laccase and Characterization of Degradation Products

“…Low-error-rate (0 to 4 mutations/kb) mutagenesis was used for introducing random mutations in the 816-bp segment of the lcc gene from C. bulleri. This segment was toward the C-terminal region and is proposed to contain the catalytic Cu centers and putative substrate-binding sites (34). The C-terminal region of both ascomycete and basidiomycete laccase is reported to influence the catalytic property of laccase (23,24).…”

Reappraising a Controversy: Formation and Role of the Azodication (ABTS2+) in the Laccase-ABTS Catalyzed Breakdown of Lignin

“…The darker red tone of the concentrate compared to the filtrate fraction suggests a relative enrichment of the red derivatives (bound in complexes?) with high-MW compounds and especially an adduct constellation with (enzyme) proteins [30,65,68,69].…”

Improving the Indigo Carmine Decolorization Ability of a Bacillus amyloliquefaciens Laccase by Site-Directed Mutagenesis

“…It has been suggested that Asp501 in B. subtilis laccase lies at the surface of the water channel by which the dioxygen molecule approaches the trinuclear copper center. It also is involved in the formation of hydrogen bonds with the solvent molecules in the channel [14]. Multiple sequence alignments showed that Asp501 has only been found in laccases from Bacillus sp., whereas a glycine residue at the corresponding position existed in other bacterial and fungal laccases ( Figure S2).…”