Proc. Natl. Acad. Sci. USA 111, 13822–13827 (2014)

Polysaccharide monooxygenases (PMOs) are Cu-dependent enzymes that cleave carbohydrate polymers including chitin, cellulose and hemicellulose. The structures of these enzymes display several conserved elements, including a Cu-coordinating 'histidine brace'. However, their sequence homology is low, making discovery of new family members difficult. Vu et al. now use the signal peptide associated with these secreted proteins along with the strict requirement for an N-terminal histidine—part of the histidine brace—to search Neurospora crassa for putative PMOs. The authors found 21 such proteins whose sequences also contained the second residue of the histidine brace and a known active site motif. NCU08746 was notable in containing a C-terminal domain related to the family 20 carbohydrate-binding module (CBM20), also known as the starch-binding domain, and so was predicted to bind amylose, a substrate not yet represented among PMOs. In vitro characterization demonstrated that NCU08746 can cleave several starch substrates. This activity is dependent on oxygen and a reductant, a role that can be played by the cellobiose dehydrogenase known to pair with cellulose-degrading PMOs, though whether this partnership occurs in vivo remains unknown. The enzyme binds and is dependent for activity on a single copper atom, and spectroscopic data support a metal binding site consistent with those of other PMOs. These results provide an important expansion of the PMO family and offer a new starch-degrading enzyme for potential industrial applications.