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)

Research output: Contribution to journalArticleScientificpeer-review

Abstract

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 β-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.IMPORTANCE 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 PQQ-dependent 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
Volume85
Issue number24
DOIs
Publication statusPublished - Dec 2019
MoE publication typeA1 Journal article-refereed

Fingerprint

PQQ Cofactor
Cytochromes b
crystal structure
quinones
cytochrome b
cytochrome
Oxidoreductases
enzyme
eukaryote
Eukaryota
Enzymes
enzymes
eukaryotic cells
prokaryote
carbohydrate
glucose
glucose dehydrogenase (pyrroloquinoline-quinone)
cellobiose-quinone oxidoreductase
Coenzymes
coenzymes

Keywords

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

Cite this

Takeda, Kouta ; Ishida, Takuya ; Yoshida, Makoto ; Samejima, Masahiro ; Ohno, Hiroyuki ; Igarashi, Kiyohiko ; Nakamura, Nobuhumi. / Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase. In: Applied and Environmental Microbiology. 2019 ; Vol. 85, No. 24.
@article{36aaa18dbfc54dbd8b4d8aa244af10fa,
title = "Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase",
abstract = "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 β-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.IMPORTANCE 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 PQQ-dependent 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.",
keywords = "AA12, AA8, Carbohydrate-Active Enzymes database, Coprinopsis cinerea, cytochrome b, pyrroloquinoline quinone",
author = "Kouta Takeda and Takuya Ishida and Makoto Yoshida and Masahiro Samejima and Hiroyuki Ohno and Kiyohiko Igarashi and Nobuhumi Nakamura",
year = "2019",
month = "12",
doi = "10.1128/AEM.01692-19",
language = "English",
volume = "85",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "24",

}

Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase. / Takeda, Kouta; Ishida, Takuya; Yoshida, Makoto; Samejima, Masahiro; Ohno, Hiroyuki; Igarashi, Kiyohiko (Corresponding Author); Nakamura, Nobuhumi (Corresponding Author).

In: Applied and Environmental Microbiology, Vol. 85, No. 24, e01692-19, 12.2019.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase

AU - Takeda, Kouta

AU - Ishida, Takuya

AU - Yoshida, Makoto

AU - Samejima, Masahiro

AU - Ohno, Hiroyuki

AU - Igarashi, Kiyohiko

AU - Nakamura, Nobuhumi

PY - 2019/12

Y1 - 2019/12

N2 - 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 β-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.IMPORTANCE 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 PQQ-dependent 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.

AB - 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 β-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.IMPORTANCE 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 PQQ-dependent 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.

KW - AA12

KW - AA8

KW - Carbohydrate-Active Enzymes database

KW - Coprinopsis cinerea

KW - cytochrome b

KW - pyrroloquinoline quinone

UR - http://www.scopus.com/inward/record.url?scp=85075813658&partnerID=8YFLogxK

U2 - 10.1128/AEM.01692-19

DO - 10.1128/AEM.01692-19

M3 - Article

C2 - 31604769

AN - SCOPUS:85075813658

VL - 85

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 24

M1 - e01692-19

ER -