Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase

Kouta Takeda, Takuya Ishida, Makoto Yoshida, Masahiro Samejima, Hiroyuki Ohno, Kiyohiko Igarashi (Corresponding Author), Nobuhumi Nakamura (Corresponding Author)

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    Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo-and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer. Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQdependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.
    Original languageEnglish
    Article numbere01692-19
    Number of pages13
    JournalApplied and Environmental Microbiology
    Issue number24
    Publication statusPublished - 27 Nov 2019
    MoE publication typeA1 Journal article-refereed


    Funding was provided by a Grant-in-Aid for Young Scientific Research (B) (grant no. 17K17703 to K.T.), and by a Grant-in-Aid for Scientific Research (B) (grant no. 15H04526 and 18H02252 to M.Y. and K.I. and grant no. 19H03013 to K.I.) from the Japan Society for the Promotion of Science (JSPS). This research was also supported by a Grant-in-Aid for Innovative Areas from the Japanese Ministry of Education, Culture, Sports, and Technology (MEXT; grant no. 18H05494 to K.I.). K.I. thanks Business Finland (BF; previously, the Finnish Funding Agency for Innovation [TEKES]) for the support of the Finland Distinguished Professor (FiDiPro) program Advanced Approaches for Enzymatic Biomass Utilization and Modification (BioAD).


    • AA12
    • AA8
    • Carbohydrate-Active Enzymes database
    • Coprinopsis cinerea
    • cytochrome b
    • pyrroloquinoline quinone


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