The impact of reductants on the catalytic efficiency of a lytic polysaccharide monooxygenase and the special role of dehydroascorbic acid

Abstract

Monocopper lytic polysaccharide monooxygenases (LPMOs) catalyse oxidative cleavage of glycosidic bonds in a reductant-dependent reaction.Recent studies indicate that LPMOs, rather than being O 2 -dependent monooxygenases, are H 2 O 2 -dependent peroxygenases. Here, we describe SscLPMO10B, a novel LPMO from the phytopathogenic bacterium Streptomyces scabies and address links between this enzyme's catalytic rate and in situ hydrogen peroxide production in the presence of ascorbic acid, gallic acid and L-cysteine. Studies of Avicel degradation showed a clear correlation between the catalytic rate of SscLPMO10B and the rate of H 2 O 2 generation in the reaction mixture. We also assessed the impact of oxidised ascorbic acid, dehydroascorbic acid (DHA), on LPMO activity, since DHA, which is not considered a reductant, was recently reported to drive LPMO reactions. Kinetic studies, combined with NMR analysis, showed that DHA is unstable and converts into multiple derivatives, some of which are redox active and can fuel the LPMO reaction by reducing the active site copper and promoting H 2 O 2 production. These results show that the apparent monooxygenase activity observed in SscLPMO10B reactions without exogenously added H 2 O 2 reflects a peroxygenase reaction.

Substrate-Dependent Cellulose Saccharification Efficiency and LPMO Activity of Cellic CTec2 and a Cellulolytic Secretome from Thermoascus aurantiacus and the Impact of H2O2-Producing Glucose Oxidase

“…The legend shown in Panel A is valid for all panels.In all experiments, the enzyme loading was 4 mg/g DM substrate. Note that 1 mM of ascorbic acid can generate more than 1 mM of LPMO product, as recently demonstrated 31. Error bars indicate standard deviations between triplicates.…”

“…In order to promote LPMO activity on lignin-poor cellulosic materials, reducing agents must be supplemented to the reactions. Studies of LPMO efficiency in reactions with various reductants have illustrated that the identity of the reductant significantly impacts LPMO activity due to the greatly varying rates of in situ H 2 O 2 generation in reactions involving the reductant and molecular O 2 . In the reactions with SPS described below, both ascorbic acid (AscA) and gallic acid (GA) were used.…”

Characterization of a Dual Cellulolytic/Xylanolytic AA9 Lytic Polysaccharide Monooxygenase from Thermothelomyces thermophilus and its Utilization Toward Nanocellulose Production in a Multi-Step Bioprocess

“…Being able to utilize both O 2 and H 2 O 2 as a co-substrate and many different compounds as electron donors, their activity as monooxygenases and peroxygenases has been debated. 15,16 They may also participate in non-specific Fenton-type reactions due to their copper-containing active site. 17 Many different LPMO genes have been found in the genome of a variety of lignocellulose-degrading microorganisms, thus raising questions about their functional properties.…”

“…LPMOs boost the activity of cellulases not only by oxidatively cleaving the glycosidic bonds of polysaccharides after a hydroxylation of the C1, C4, or both C1/C4 carbon atoms and create new nick points, but also, in case of enzymes with C1 and C1/C4-regioselectivity, by adding surface charge and leading to defibrillation. , The enigmatic nature of LPMOs has been the subject of extensive study and it has not been completely understood yet. Being able to utilize both O 2 and H 2 O 2 as a co-substrate and many different compounds as electron donors, their activity as monooxygenases and peroxygenases has been debated. , They may also participate in non-specific Fenton-type reactions due to their copper-containing active site …”

Mono-oxigenase lítica de polissacarídeos TtLPMO9H: caracterização e mecanismos moleculares de ativação catalítica

“…Outro fator que pode influenciar a atividade da LPMO é o H2O2 presente na reação, formado tanto pela LPMO que foi reduzida, mas não se ligou ao substrato, 62 quanto por reações entre o agente redutor e íons cobre livres em solução. Ácido ascórbico é especialmente reativo 136 e resulta em quantidades de H2O2 que são suficientes tanto para a atividade da LPMO quanto para sua inativação.…”