Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass

Abstract

Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol: quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.A uxiliary activities (AA) are a widespread group of catalytic modules involved in plant cell wall degradation. They were first introduced in 2013 into the carbohydrate-active enzymes (CAZy [www.cazy.org]) database (1). This AA class encompasses and extends an earlier classification dedicated to fungal ligninolytic enzymes and comprises 10 families, some of which have subfamilies (2). The AA family group enzymes possess the potential ability to help the original carbohydrate-active enzymes gain access to the carbohydrates embedded in the plant cell wall. Lignin, the main noncarbohydrate structural component of plant cell walls, consists of an intricate network of aromatic compounds forming a strong, hydrophobic, insoluble barrier closing access to the carbohydrates. Microorganisms have therefore set up different strategies to either modify or degrade lignin as a first step to gain access to the carbohydrate moiety and convert it into an energy source (3).In this new AA classification, family AA3 belongs to the glucose-methanol-choline (GMC) oxidoreductase family first defined by Cavener (4). AA3 enzymes are flavoproteins containing a flavin adenine dinucleotide (FAD)-binding domai...

Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

“…Among them, AAQO1 was considered as a strict dehydrogenase since it was unable to transfer electrons to molecular oxygen while AAQO2 and AAQO3 were considered as dehydrogenases with some residual oxidase activity27. On the other hand the AAO from U. maydis was able to efficiently oxidize anisyl alcohol using oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol as electron acceptors28.…”

“…AAQO1 (scf185002.g8), AAQO2 (scf184746.g7) and GDH (scf184803.g17) from P. cinnabarinus were produced in A. niger as described in 27 and29. All of the enzymes were purified to homogeneity and quantified as described previously.…”

Oxidoreductases and Reactive Oxygen Species in Conversion of Lignocellulosic Biomass

“…Red stars indicate the absence of kinetic data. Abbreviations (and associated references for corresponding data) are as follows: Lac, laccase(380)(381)(382)(383); POD, peroxidase(380,(384)(385)(386)(387)(388); CDH, cellobiose dehydrogenase(102,(128)(129)(130)(389)(390)(391)(392); GOX, glucose oxidase(167,(393)(394)(395)(396)(397)(398); AAO, aryl alcohol oxidase(166,399,400); GDH, glucose dehydrogenase(401,402); AAQO, aryl alcohol quinone oxidoreductase(403); PDH, pyranose dehydrogenase(404)(405)(406)(407); AOX, alcohol oxidase(177,(408)(409)(410); P2O, pyranose 2-oxidase(181,(411)(412)(413); VAO, vanillyl alcohol oxidase(186,187); GLOX, glyoxal oxidase(170,<...>…”

iTRAQ-Based Proteomic Analysis Reveals the Mechanisms of Botrytis cinerea controlled with Wuyiencin

“…On the other hand, almost half of the PRM validated, down-regulated proteins identified in this study display oxidoreductase activity. While fungal oxidoreductases play a role in a wide array of processes, several are known to be involved in biomass degradation, mediating electron transfer for cellular respiration, and even participating in the generation of free radicals [28]. As shown in Table 1 Figure 6).…”