SirexAA-E is a highly cellulolytic Streptomyces associated with the invasive pine-boring woodwasp Sirex noctilio. When grown on cellulose as the sole carbon source, SirexAA-E secretes a host of hydrolytic and oxidative enzymes that work synergistically to deconstruct cellulosic substrates. The most abundantly secreted enzymes include a putative endolytic Cu dependent polysaccharide monooxygenase (or PMO, termed SactE_3159) and two exocellulases presumed to be reducing-end and non-reducing end glycosyl hydrolases from GH families 6 and 48. These three enzymes must work synergistically to achieve efficient conversion of cellulosic substrates into cellobiose, which is the main growth substrate for SirexAA-E. We show here that SactE_0236 and SactE_0237 are processive cellobiohydrolases whose catalytic activity is enhanced in the presence of SactE_3159. We confirm that SactE_3159 contains a redox-cycling mononuclear Cu(II) site and consumes O2 and reducing equivalents to achieve an endolytic, oxidative cleavage of cellulosic substrates. The product profile for SactE_3159 includes an aldonic acid ladder, suggesting SactE_3159 reacts at the C1 carbon of the �(1?4) glycosidic linkage in order to cleave this bond. A stead-state kinetic analysis of the catalytic synergy between SactE_3159, SactE_0236, and SactE_0237 will be presented along with additional investigations of the properties and reactivity of SactE_3159.

Kinetic and mechanistic studies with polysaccharide monooxygenase from SirexAA-E

248th National Meeting (2014)

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