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Trichoderma reesei Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization.

ABSTRACT: Pyrroloquinoline quinone (PQQ) is an ortho-quinone cofactor of several prokaryotic oxidases. Widely available in the diet and necessary for the correct growth of mice, PQQ has been suspected to be a vitamin for eukaryotes. However, no PQQ-dependent eukaryotic enzyme had been identified to use the PQQ until 2014, when a basidiomycete enzyme catalyzing saccharide dehydrogenation using PQQ as a cofactor was characterized and served to define auxiliary activity family 12 (AA12). Here we report the biochemical characterization of the AA12 enzyme encoded by the genome of the ascomycete Trichoderma reesei (TrAA12). Surprisingly, only weak activity against uncommon carbohydrates like l-fucose or d-arabinose was measured. The three-dimensional structure of TrAA12 reveals important similarities with bacterial soluble glucose dehydrogenases (sGDH). The enzymatic characterization and the structure solved in the presence of calcium confirm the importance of this ion in catalysis, as observed for sGDH. The structural characterization of TrAA12 was completed by modeling PQQ and l-fucose in the enzyme active site. Based on these results, the AA12 family of enzymes is likely to have a catalytic mechanism close to that of bacterial sGDH.IMPORTANCE Pyrroloquinoline quinone (PQQ) is an important cofactor synthesized by prokaryotes and involved in enzymatic alcohol and sugar oxidation. In eukaryotes, the benefit of PQQ as a vitamin has been suggested but never proved. Recently, the first eukaryotic enzyme using PQQ was characterized in the basidiomycete Coprinopsis cinerea, demonstrating that fungi are able to use PQQ as an enzyme cofactor. This discovery led to the classification of the fungal PQQ-dependent enzymes in auxiliary activity family 12 (AA12) of the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org) classification. In the present paper, we report on the characterization of the ascomycete AA12 enzyme from Trichoderma reesei (TrAA12). Our enzymatic and phylogenetic results show divergence with the only other member of the family characterized, that from the basidiomycete Coprinopsis cinerea The crystallographic structure of TrAA12 shows similarities to the global active-site architecture of bacterial glucose dehydrogenases, suggesting a common evolution between the two families.

SUBMITTER: Turbe-Doan A

PROVIDER: S-EPMC6881810 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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<i>Trichoderma reesei</i> Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization.

Turbe-Doan Annick A Record Eric E Lombard Vincent V Kumar Rajender R Levasseur Anthony A Henrissat Bernard B Garron Marie-Line ML

Applied and environmental microbiology 20191127 24

Pyrroloquinoline quinone (PQQ) is an <i>ortho</i>-quinone cofactor of several prokaryotic oxidases. Widely available in the diet and necessary for the correct growth of mice, PQQ has been suspected to be a vitamin for eukaryotes. However, no PQQ-dependent eukaryotic enzyme had been identified to use the PQQ until 2014, when a basidiomycete enzyme catalyzing saccharide dehydrogenation using PQQ as a cofactor was characterized and served to define auxiliary activity family 12 (AA12). Here we repor ...[more]

PMID: 31604773

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Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-?-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Project description:Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from Coprinopsis cinerea, CcPDH, the founding member of the recently discovered auxiliary activity family AA12. CcPDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that CcPDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F (NcLPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C (NcLPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested CcPDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from CcPDH had a considerable negative impact on the efficiency of the CcPDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs.IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family, CcPDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individual CcPDH domains (a dehydrogenase, a cytochrome, and a carbohydrate-binding domain), (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of the CcPDH-LPMO system.

Project description:Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La3+). ExaF was purified from cells grown with both calcium (Ca2+) and La3+ and with Ca2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La3+ over Ca2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca2+-plus-La3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca2+-only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La3+ is present, expanding the role of lanthanides to multicarbon metabolism.ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases.

Project description: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.

Dataset Information

Trichoderma reesei Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization.

Publications

<i>Trichoderma reesei</i> Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization.

Similar Datasets

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