Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus Phanerochaete chrysosporium produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal b-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of P. chrysosporium cellobiose dehydrogenase. The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a β-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a b-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer. The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl δ and ε nitrogens, rather than the usual N-ε/N-εligation. This is the first example of a bis-His N-ε/N-δ coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of α/β-type and a substrate-binding subdomain consisting of a seven-stranded β-sheet and six α-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-2701 |
| Date | January 2002 |
| Creators | Hällberg, Martin |
| Publisher | Uppsala universitet, Institutionen för cell- och molekylärbiologi, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1104-232X ; 767 |
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