Although PQQ has nutritional importance and pharmacological

Although PQQ has nutritional importance and pharmacological effects in higher organisms, including humans [15], until recently, there was no clear evidence for the use of PQQ as a co-factor in a eukaryotic enzyme. This review presents a novel eukaryotic PQQ-dependent quinohemoprotein recently discovered from the saprophytic basidiomycete Coprinopsis cinerea, with a substrate specificity not previously described for fungi, and with possible links to fungal biomass conversion.
A new group of PQQ-dependent enzymes Cellulolytic fungi are important decomposers of plant biomass and secrete not only a set of glycoside hydrolases but also numerous oxidoreductases, including cellobiose dehydrogenases (CDHs) and lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant cell wall polysaccharides [16,]. In the database of Carbohydrate-Active enZymes (CAZy, http://www.cazy.org/), these oxidoreductases are categorized into Auxiliary Activities (AAs) families, which comprise redox enzymes that act in conjunction with other CAZymes [18]. Family AA12 was established in September 2014 upon the discovery of a secreted PQQ-dependent pyranose dehydrogenase, derived from C. cinerea (CcPDH). CcPDH is composed of an N-terminal cytochrome domain belonging to the AA8 family, the AA12 catalytic domain and a C-terminal cellulose-binding domain belonging to carbohydrate-binding modules (CBMs) family 1, as shown in Figure 2 []. The existence of CBM1 indicated the role of this enzyme in plant cell wall degradation, and thus led the dehydrogenase domain of CcPDH to be introduced into AA class as a new family. The recombinant protein heterologously expressed in Pichia pastoris showed oxidative activity towards various sugars (see below) in the presence of PQQ and calcium ion. CcPDH is the first PQQ-dependent enzyme found in eukaryotes. BLAST searches showed that the amino 50 dub synthesis sequence of the AA12 domain in CcPDH is not similar with those of known prokaryotic PQQ-dependent enzymes, indicating that AA12 enzymes indeed represent a new family of quinoproteins. Genes encoding homologues of the PQQ-binding dehydrogenase domain of CcPDH are found in many fungal genomes. The fungal AA12 homologues show four types of domain structures, single AA12 domain (majority), AA12-CBM1, AA8-AA12, and AA8-AA12-CBM1 signal peptide, as shown in Figure 3. Interestingly, AA12 homologues were also found in bacterial, archaeal, and amoebozoal genomes, and all these enzymes show single domain structures []. The fact that non-fungal AA12 homologues are also PQQ-dependent enzymes was confirmed by the study of an AA12-homologue from the bacterium Pseudomonas aureofaciens (named Pa2KGDH) []. This study showed that Pa2KGDH specifically oxidizes the C1-position of 2-keto-d-glucose (2KG; also known as d-glucosone), and only in the presence of PQQ, producing 2-keto-d-gluconic acid (2KGA), an activity that is also displayed by CcPDH, as outlined below. This indicates that PQQ-dependent AA12 enzymes, with primary sequences that are remarkably distinct from those of previously characterized prokaryotic PQQ-dependent enzymes, are widely distributed, from prokaryotes to eukaryotes.
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.