Cellobiose Dehydrogenase - wild type and its variants (addition of bivalent 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 revealed the role of two amino acids (M309 and R698) located in the domain interface which are hypothesized to limit interdomain electron transfer. CDH from Myriococcum thermophilum was used for site-specific mutagenesis. The wild-type and all variants (M309A, R698S and M309A/R698S) were analyzed kinetically by stopped-flow studies and structurally by using small-angle X-ray scattering. We showed an increase of CDH flexibility at higher pH, indicating alterations of domain mobility.

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 Na- acetate buffer pH 5.5 and a HEPES pH 6.5 buffer with and without CaCl2 (50 and 100 mM) 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.

MtCDH.. are the files for the wild-type; M309A..are the files for the M309A variant; R698S..are the files for the R698S variant; M309AR698S..are the files for the double mutant variant

2023-04-06

2023-04-08

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