Abstract
Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.
Highlights
Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown
The L3 loop shows higher sequence variability, and both the L3 and the LC loops are somewhat shorter in NcLPMO9A and NcLPMO9D compared with NcLPMO9C
Previous NMR studies on NcLPMO9C have shown that His-1, Ala-80, His-83, and His-155 (Fig. 1A, marked with * at positions 1, 78, 81, and 153, respectively) are involved in binding of both oligocellulose and polymeric xyloglucan [46], and three of these four residues are conserved among the three NcLPMOs
Summary
The three-dimensional structure of NcLPMO9A with copper bound was solved to 1.6-Å resolution (Table S1) and revealed a typical LPMO structure, both with regard to the overall fold and the copper-containing active site (Fig. 1). Product analysis after incubation of NcLPMO9A with phosphoric acid–swollen cellulose (PASC) using chromatographic methods [44], MS/MS analysis [42], and identification of characteristic products obtained upon reduction [33] showed that NcLPMO9A exclusively oxidizes C4, confirming results of Vu et al [38] (results not shown)
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