Dataset Information

Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

ABSTRACT: Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o-diphenols to o-quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l-tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a library of 39?mutants of AUS from Coreopsis grandiflora (CgAUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA₂ , HisB₁ , and HisB₂ ) is tuned by their adjacent residues (HisB₁ +1, HisB₂ +1, and waterkeeper residue) either to react as stronger bases or / and to stabilize a position permissive for substrate proton shuffling. This provides the understanding for C-H activation based on the type-III copper center to be used in future biotechnological processes.

SUBMITTER: Kampatsikas I

PROVIDER: S-EPMC7693034 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

Kampatsikas Ioannis I Pretzler Matthias M Rompel Annette A

Angewandte Chemie (International ed. in English) 20200909 47

Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o-diphenols to o-quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l-tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a libr ...[more]

PMID: 32701181

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Project description:BackgroundNADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.ResultsWe assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.ConclusionThis report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

Dataset Information

Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

Publications

Identification of Amino Acid Residues Responsible for C-H Activation in Type-III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase.

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