Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity

Abstract

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1-and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the β-(1!4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan-and oligosaccharideactive AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

Comparison of Six Lytic Polysaccharide Monooxygenases from Thermothielavioides terrestris Shows That Functional Variation Underlies the Multiplicity of LPMO Genes in Filamentous Fungi

“…Most of the products in reactions with Tt LPMO9E contained multiples of three pentose units (3 n ), whereas Tt LPMO9A generated a range of XG-oligosaccharides with more even distribution of numbers of pentose units. This suggests that Tt LPMO9E is a little tolerant to substitution adjacent to the scissile bond, while Tt LPMO9A is tolerant to substitution adjacent to the scissile bond, a type of action that has been described for C 1 /C 4 -oxidizing LPMOs ( 69 ). Notably, the most dominant species were the oxidized species for Tt LPMO9E and the native species for Tt LPMO9A (which were labeled accordingly in Fig.…”

“…Xyloglucan-active LPMO9s have been studied in quite some detail, and several authors have tried to link sequence features of the LPMOs (in particular the presence and length of certain loop regions) to observed XG activity and the impact of substitutions ( 69 , 71 , 72 ). Interestingly, the observed activities of Tt LPMO9A and Tt LPMO9E do not match with these previous classifications, which would have predicted these two LPMOs to be inactive on TXG, or, when active, to both be substitution tolerant.…”

Quantifying Oxidation of Cellulose-Associated Glucuronoxylan by Two Lytic Polysaccharide Monooxygenases from Neurospora crassa

“…The availability of at least one crystal structure for each of the clades with xylan-active LPMOs and the availability of crystal structures for LPMOs found not to be active on glucuronoxylan provide an opportunity to assess possible structural determinants of xylanolytic activity. Despite some recent progress ( 50 – 52 ), the structural determinants of LPMO substrate specificity remain largely unknown. The substrate-binding surfaces of LPMOs vary considerably (Fig.…”

Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

“…In another study on Aspergillus nidulans , the gene encoding AN1602 ( 44 ) (an AA9-CBM1 protein displaying 65% sequence identity with Ao AA9 and Af AA9) was highly transcribed during growth on cellulose and xylan, and the enzyme was shown to be active on both cell6 and XG ( 44 , 45 ). Recently, XG and cell6 activities have also been reported for an AA9-CBM1 LPMO from Aspergillus tamarii ( At AA9A) for which the catalytic AA9 domain shares about 80% sequence identity with Ao AA9A and Af AA9C ( 46 ).…”