br The PQQ and heme

The PQQ and heme b cofactors of CcPDH are located in the 45 kDa AA12 and the 21 kDa AA8-cytochrome domains, respectively, and these domains are connected by a proline-rich linker region. The domain organization of CcPDH resembles those of some CDHs, which have a flavin-containing AA3 domain instead of the PQQ-containing AA12 domain (Figure 2). This domain organization is unique for PQQ enzymes, as only a c-type cytochrome domain and membrane-binding domain have been identified in known PQQ enzymes so far. CcPDH showed PQQ-dependent catalytic activity, as demonstrated by the increase in activity observed upon increasing the PQQ concentration up to stoichiometric amounts. Isothermal titration calorimetry data demonstrated that the AA12 domain binds PQQ with a 1:1 stoichiometry, and has strong affinity for the co-factor, with a dissociation constant, Kd, of 1.1 nM []. Most recently, we determined the crystal structure of the AA12 domain of CcPDH, which provides structural evidence for binding of PQQ to the protein (Takeda et al., unpublished results). The structure shows that PQQ is bound at the active site together with a catalytically essential calcium ion. The co-occurrence of the AA12 and the AA8 domains in CcPDH would allow for TMP269 transfer between these domains. Such electron transfer is known to occur in CDH, and the AA8 family in CAZy is in fact largely comprised of cytochrome domains of multi-domain CDHs. One notable exception concerns the carbohydrate-binding cytochrome b562 from Phanerochaete chrysosporium, a two-domain structure consisting of an AA8 and a CBM1 but not containing a dehydrogenase domain [22]. AA8 domains contain a 6-coordinated low-spin heme b group within an ellipsoidal antiparallel β-sandwich fold [23]. The heme is bound in a hydrophobic pocket at one face, with one heme edge being exposed to the solvent, and being axially ligated by Met and His. The amino acid sequence of the AA8 domain in CcPDH has 32–42% sequence identity with the AA8 domains of basidiomycete CDHs. The UV–vis and resonance Raman spectra of CcPDH in the oxidized and reduced forms are in good agreement with spectra obtained for the AA8 domain of P. chrysosporium CDH (PcCDH) []. Our electrochemical studies indicated that the redox potential of the heme is around +130 mV versus NHE at pH 7.0, which is almost identical to the value obtained for PcCDH []. Plant cell wall degrading enzymes often contain a non-catalytic CBM. The majority of CBMs attached to fungal cellulolytic enzymes belongs to family 1. The presence of a CBM1 indicates a role in plant cell wall degradation, as the main function of CBM1s is to facilitate binding of the enzyme to the surface of cellulose, which is thought to enhance catalytic efficiency by increasing the effective enzyme concentration near the substrate. Some ascomycetous CDHs possess a CBM1 at their C-terminus, whereas basidiomycetous CDHs lack this domain [24]. The C-terminal CBM1 of CcPDH contains three conserved aromatic residues that likely contribute to binding on cellulose, as suggested by in-depth studies of the functionality of other CBM1 domains [16]. Because CcPDH shows high affinity toward microcrystalline and amorphous celluloses [], in its natural environment, the enzyme presumably is localized on the surface of cellulose.