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

Alfons K G Felice,Christoph Sygmund,Roland Ludwig,Christian Schuster,Stefan Scheiblbrandner,Frantisek Filandr,Lorenz Schwaiger,Petr Halada,Alan Kadek,Daniel Kracher,Christophe V F P Laurent,Franziska Schachinger,Petr Man,Chris Oostenbrink

doi:10.1021/acscatal.0c05294

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.

Highlights

The catalytic activity of lytic polysaccharide monooxygenase (LPMO) and its interaction with cellobiose dehydrogenase (CDH) have been reported to increase the rate of cellulose hydrolysis from the recalcitrant biomass and to increase the overall efficiency of enzymatic cocktails.[1−5] In contrast to electron-donating, low-molecular weight reductants of LPMO such as gallate or ascorbate, CDH is specific for LPMO and shows a fast electron transfer at physiological concentrations.[6,7] CDH is an extracellular flavocytochrome and contains FAD and a b-type heme in the flavodehydrogenase (DH) and cytochrome (CYT) domains, respectively, which are connected via a flexible linker
We evaluated the structural and kinetic determinants of the domain interaction to test recent results obtained by Courtade et al, who showed the binding of CDH and CYT to the LPMO active site by means of 15N-HSQC and 13C-aromatic-HSQC,[15] and by Laurent et al who modeled the interaction between both enzymes.[16]
The end of the N-terminal CYT domain is defined by a cysteine residue forming a disulfide bond (CYTA: Q1−C211, CYTB: Q1−C216, for brevity, we denote the domains and the linker of NcCDHIIA by A and NcCDHIIB by B)

Summary

■ INTRODUCTION

The catalytic activity of lytic polysaccharide monooxygenase (LPMO) and its interaction with cellobiose dehydrogenase (CDH) have been reported to increase the rate of cellulose hydrolysis from the recalcitrant biomass and to increase the overall efficiency of enzymatic cocktails.[1−5] In contrast to electron-donating, low-molecular weight reductants of LPMO such as gallate or ascorbate, CDH is specific for LPMO and shows a fast electron transfer at physiological concentrations.[6,7] CDH is an extracellular flavocytochrome and contains FAD and a b-type heme in the flavodehydrogenase (DH) and cytochrome (CYT) domains, respectively, which are connected via a flexible linker. With IDET depending on the closed state or at least very close proximity between DH and CYT, a steric mismatch between the domain surfaces, repulsive electrostatic interactions, or a linker that provides too much mobility will reduce IDET This is supported by the inspection of the kobs[563] rates for both evolved wild-type CDHs and the chimeric CDHs (Table 2). Mixed factor principal component analysis (PCA), including the quantitative variables (kcat, kobs, IDET, IPET, CYT midpoint redox potential, and glycosylation) and qualitative variables [DH-, CYT-, and linker-type (Tables S5 and Figure S9)], has been performed on the data set from wild-type and chimeric CDHs to explore intercorrelation. Glycosylation shows the smallest effect of all quantitative variables, while the kinetic variables cluster as expected from bivariate analysis

■ DISCUSSION

■ ACKNOWLEDGMENTS

■ REFERENCES

Electrostatics of Nanosystems