Cellobiose Dehydrogenase (addition of divalent cations)

The interdomain electron transfer between the catalytic flavodehydrogenase domain and the electron transferring cytochrome domain of cellobiose dehydrogenase plays an essential role in biocatalysis, biosensors, and biofuel cells as well as for its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domain of cellobiose dehydrogenase (CDH), which are hypothesized to limit interdomain electron transfer in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum was used to study the CDH mobility in the presence of divalent cations. We showed that the presence of calcium ions decreases the mobility of the cytochrome domain.

The sample used for the experiments represents MtCDH IIA: gene cdh, UniProt A9XK88, the CYT domain belongs to CAZy family AA8, and the DH domain to CAZy family AA3_1. SEC-SAXS data were collected at the SIBYLS beamline 12.3.1 at Advanced Light Source (LBNL Berkeley, California, USA) at 1.27 Å wavelength with a Pilatus 2M detector 2.1 m sample-to-detector distance, corresponding to a q range from 0.01 to 0.4 Å-1. The scattering vector is defined as q = 4π sinθ/λ, where 2θ is the scattering angle, and λ is the X-ray wavelength. For the measurements a 50 mM HEPES pH6.5 buffer with and without CaCl2 was used. A size-exclusion chromatography was directly coupled with a SAXS flow cell and a multi-angle light scattering (SEC-SAXS-MALS). For analyzing the data and computing the pair distance distribution function the program SCÅTTER (https://bl1231.als.lbl.gov/scatter/) was used. The SAXS frames recorded before the protein elution was used for the background subtraction.

gMtCDH.. are the files for the glycosylated (native) CDH. dgMtCDH.. are the files for the deglycosylated CDH

2023-02-28

2023-03-14

SEC-SAXS

Advanced Light Source

SIBYLS BL12.3.1

1.27 Å

2.1 m

Sample:

• Macromolecule 1: MtCDH
• Sample Full Name: Cellobiose dehydrogenase
• Sample Type: Protein
• Source Organism: Myriococcum thermophilum
• Source Organism NCBI Taxonomy ID: 2588717
• Expression System: Pichia pastoris X-33
• Expression NCBI Taxonomy ID: 4922
• Uniprot ID: A9XK88
• Sequence or Chemical Formula:
• QNNVPNTFTDPDSGITFNTWGLDEDSPQTQGGFTFGVALPSDALTTDASEFIGYLKCARNDESGWCGISLGGPMTNSLLITAWPHEDTVYTSLRFATGYAMPDVYEGDAEITQVSSSVNSTHFSLIFRCKNCLQWSHGGSSGGASTSGGVLVLGWVQAFDDPGNPTCPEQITLQQHDNGMGIWGAQLNTDAASPSYTDWAAQATKTVTGDCEGPTETSVVGVPVPTGVSFDYIVVGGGAGGIPAADKLSEAGKSVLLIEKGFASTANTGGTLGPEWLEGHDLTRFDVPGLCNQIWVDSKGIACEDTDQMAGCVLGGGTAVNAGLWFKPYSLDWDYLFPDGWKYNDVQPAINRALSRIPGTDAPSTDGKRYYQEGFEVLSKGLAAGGWTSVTANNAPDKKNRTFAHAPFMFAGGERNGPLGTYFQTAKKRNNFDVWLNTSVKRVIREGGHITGVEVEPFRDGGYEGIVPVTKVTGRVILSAGTFGSAKILLRSGIGPEDQLEVVAASEKDGPTMIGNSSWINLPVGYNLDDHLNTDTVISHPDVVFYDFYEAWDDPIESDKNSYLESRTGILAQAAPNIGPMFWEEIVGADGIVRQLQWTARVEGSLGAPNGHTMTMSQYLGRGATSRGRMTITPSLTTIVSDVPYLKDPNDKEAVIQGIINLQNALQNVANLTWLFPNSTITPREYVESMVVSPSNRRSNHWMGTNKLGTDDGRKGGSAVVDLDTRVYGTDNLFVIDASIFPGVPTTNPTSYIVVAAEHASSRILALPDLEPVPKYGQCGGREWTGSFVCADGSTCEYQNEWYSQCL