Chimeric Cellobiose Dehydrogenases Reveal the Function of Cytochrome Domain Mobility for the Electron Transfer to Lytic Polysaccharide Monooxygenase

Abstract

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus’ own LPMOs (NcLPMO9C and NcLPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein–protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme b cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 105 and 1.1 × 106 M–1 s–1.

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications

“…CDH oxidizes various sugars, including cellobiose or lactose and in some instances even glucose, at its DH while concurrently electrons are transferred to FAD. Reoxidation of FADH 2 can occur directly by reduction of various quinones as electron acceptors, or by interdomain electron transfer (IET) to the heme group, from where they can be passed on to cytochrome c as an artificial electron acceptor or to lytic polysaccharide monooxygenases (LPMO), the presumed natural interaction partner. − Thus, CDH and LPMO are part of an extracellular electron transfer system efficiently fueling the breakdown of recalcitrant lignocellulose by sequential transfer of electrons from soluble sugars via FAD and heme b to the active site copper in LPMO …”

Unique Lysine-Rich Sequence on the CYT Domain of AfCDH Enhances Its Interdomain Electron Transfer and Activation of AA9 LPMOs

“…The oxidative system involving CDHs and copper-dependent LPMOs is crucial for effective degradation of recalcitrant polysaccharides like cellulose, , hemicellulose, starch, and chitin . Compared to using the chemical compounds (e.g., ascorbate) as electron donors, it was considered that employing an enzyme as an electron donor is advantageous because this enables a kinetically controlled supply of electrons to the LPMO. ,,, Despite the fact that the role and significance of LPMOs in the CDH-LPMO system are well recognized and much progress has been achieved in discovering and engineering LPMOs for their industrial application in commercial cellulase cocktail, − there is still less understanding about the CDH-driven reduction mechanism due to the modular structure of CDHs and the long-range electron-transfer process involving IDET and IPET in driving the LPMO reaction. , The IDET between DH and CYT domains in CDH is a particular important process controlling the overall electron-transfer rate; however, the intrinsic determinants in CDH such as the specific sequence and structural element governing the IDET remain elusive, which partially impedes our effort in engineering CDH and ameliorating the CDH-LPMO system efficiency. Here, we demonstrated that an extra lysine-rich sequence in the vicinity of heme b on the CYT domain of Af CDH played a vital role in IDET and activation of AA9 LPMOs.…”

“…Later, the structural study on the domain interaction in CDH confirmed that IDET was modulated by surface electrostatics . Recently, research on the chimeric CDHs by exchanging CYT and linkers of Neurospora crassa NcCDHIIA and Nc CDHIIB revealed that the IDET depended on steric and electrostatic interface complementarity and the length of the linker between two domains instead of the redox potential . Besides, a highly conserved tyrosine residue in the vicinity of heme b plays a crucial role in the electrostatic network between CYT and DH, which is essential for productive IDET .…”

“…It is clearly evident that the oxidative system involving CDH and LPMO in filamentous fungi is crucial for their oxidative attack on polysaccharides; nevertheless, the system validity relies on the LPMO-reducing efficiency via CDH-driven extracellular electron transfer . Compared to the IPET between CYT in CDH and LPMO, however, the IDET from DH to CYT in CDH is regarded as a limiting step for the overall CDH-driven extracellular electron transfer for the LPMO reaction, , so exploring the intrinsic factors influencing the intramolecular association between DH and CYT is vital for improving the efficiency of IDET in CDH and CDH-driven LPMO reaction systems. Since IDET is enabled by the heme propionate group A and needs a closed CDH state, it was previously presumed that the IDET rate might be governed by the redox potential difference between the FAD and heme b cofactors .…”

Deletion of AA9 Lytic Polysaccharide Monooxygenases Impacts A. nidulans Secretome and Growth on Lignocellulose

“…Electrons to reduce LPMOs can be provided by various sources such as small chemical compounds (e.g., ascorbate and monophenols) or enzymes, including the AA3_1 CDHs ( 17 ). Fungal CDHs have been shown to provide electrons for the redox-mediated oxidative cleavage of cellulose ( 18 , 19 ), while also being involved in lignin degradation ( 20 , 21 ).…”