Crystal structures of a dodecameric multicopper oxidase from Marinithermus hydrothermalis

Abstract

Multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.

Structure–function studies of a novel laccase-like multicopper oxidase fromThermothelomyces thermophilaprovide insights into its biological role

“…Asco-laccases regularly form dimeric assemblies in a head-to-head arrangement of the T1 pockets of the enzyme (Ernst et al, 2018). Moreover, a dodecameric arrangement of a bacterial MCO in its active form has recently been reported (Paavola et al, 2021). Analysis of the TtLMCO1 structure by the Protein Interfaces, Surfaces and Assemblies (PISA) server (Krissinel & Henrick, 2007) did not suggest dimer formation, while the interactions between TtLMCO1 monomers are a result of crystal packing.…”

“…To date, 77 biochemically characterized enzymes have been reported in the CAZy database, of which 25 have experimentally determined structures. Most of the known laccase structures originate from basidiomycetes and several structures are from plant (Xie et al, 2020) and bacterial (Enguita et al, 2003;Paavola et al, 2021;Olmeda et al, 2021) sources, while only five structures are been reported from ascomycetes.…”

Unusual Oligomeric Laccase-like Oxidases from Ascomycete Curvularia geniculata VKM F-3561 Polymerizing Phenylpropanoids and Phenolic Compounds under Neutral Environmental Conditions

“…Later, X-ray diffraction analysis also showed the presence of two oligomeric forms of this enzyme: dimeric and hexameric [62]. Another multicopper oxidase MhMCO from the thermophilic bacterium Marinithermus hydrothermalis with laccase activity was crystallized as a dodecamer consisting of four trimers forming a closed spherical structure [63].…”