C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases

Abstract

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanized new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyze a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

The crystal structure of CbpD clarifies substrate-specificity motifs in chitin-active lytic polysaccharide monooxygenases

“…What remains unclear, however, is whether LPMOs may have reducing-agent specificity in addition to substrate specificity (Frommhagen et al, 2016;Meier et al, 2018). Askarian and coworkers demonstrated that CbpD chitinolytic activity was enhanced by ascorbate and the redox-active compound pyocyanin, which is secreted by P. aeruginosa (Askarian et al, 2021), and Branch and coworkers demonstrated that a c-type cytochrome can activate the celluloseactive CjAA10B in Cellvibrio japonicus (Branch et al, 2021). CbpD and other LPMOs may favor reductants that are secreted by their hosts.…”

Selectivity and stability of N-terminal targeting protein modification chemistries

“…To probe this hypothesis, we studied the modification of CjX183-D, a naturally occurring cytochrome from Cellvibrio japonicus which does not contain any lysine residues. 22 N-terminal selectivity was compared to the mutant CjX183-D R51K, in which a single lysine had been installed (Fig. 3).…”

Revisiting the role of electron donors in lytic polysaccharide monooxygenase biochemistry

“…A single X158 has been characterised to date but it, as well as many other distantly-related domains, have been purified with the well-known redox molecule ubiquinone-8 bound, as revealed by their crystal structures [ 80 ]. In addition, Cbp2D is also predicted to contain cytochrome-like domains at the C-terminus of the protein, which would suggest a likely electron-transfer function, hence, an interest in them as potential LPMO electron donors [ 78 , 81 ]. In our own recent work, we were able to isolate one of the cytochrome-like domains ( Cj X183D) from Cbp2D and showed that it was likely an electron transferring, as opposed to catalytic, c -type cytochrome [ 81 ].…”

“…In addition, Cbp2D is also predicted to contain cytochrome-like domains at the C-terminus of the protein, which would suggest a likely electron-transfer function, hence, an interest in them as potential LPMO electron donors [ 78 , 81 ]. In our own recent work, we were able to isolate one of the cytochrome-like domains ( Cj X183D) from Cbp2D and showed that it was likely an electron transferring, as opposed to catalytic, c -type cytochrome [ 81 ]. Its reduced state was able to drive LPMO catalysis in the presence of O 2 but activity in the presence of H 2 O 2 was not considered at the time.…”