Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27

Okazawa, Yuka; Miyazaki, Takatsugu; Yokoi, G.; Ishizaki, Yuichi; Nishikawa, Atsushi; Tonozuka, Takashi

doi:10.1074/jbc.m115.680942

Cited by 22 publications

(22 citation statements)

References 51 publications

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“…1). The N-terminal catalytic domain consists of a five-bladed b-propeller (Gln21-Gly325), as in other GH clan-F enzymes, and the C-terminal domain (PcCBM35) takes a β-jellyroll fold (Thr326-Tyr448) structure, as in previously reported CBM35s (16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

Section: Overall Structure Of Pc13gal43amentioning

confidence: 81%

“…The CBM35 module is composed of approximately 140 amino acids. This family includes modules with various binding characteristics, and decorated with xylans, mannans, β-1,3-galactans, and glucans (16)(17)(18)(19)(20)(21). The family members are divided into four clusters based on their sequences and binding specificities (17).…”

Section: Introductionmentioning

confidence: 99%

“…The family members are divided into four clusters based on their sequences and binding specificities (17). The structures of CBM35s binding with xylan, mannan, and glucan have already been solved (16)(17)(18)(19)(20)(21), but no structure of β-1,3-galactan-binding CBM35 has yet been reported.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

Matsuyama

Kishine

Fujimoto

et al. 2020

Preprint

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Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

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Section: Overall Structure Of Pc13gal43amentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

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Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

Matsuyama

Kishine

Fujimoto

et al. 2020

Preprint

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“…These genes provide valuable resources for screening AvrLr19 candidates and pathogenesis-related genes. Among them, PTTG-01075 and PTTG-11895 are annotated as clan D glycoside hydrolases (GH-D), which are commonly referred to as plant cell wall degrading enzymes (PCWDEs) [32]. PTTG-12205 is annotated as a Major Facilitator Superfamily (MFS) of sugar transporters, which facilitates the transport of diverse molecules like sugars, vitamins, amino acids, hormones, etc.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome Analysis Based on Lr19-Virulent Mutants Strain Provides Clues for the Pathogenicity-Related Genes and Effectors of Puccinia Triticina

Fan

Wang

et al. 2020

Preprint

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Background: Wheat leaf rust caused by Puccinia triticina (Pt) remains one of the most destructive diseases of common wheat worldwide. Cultivating resistant cultivars is an effective way to control this disease, but race-specific resistance can be overcome quickly due to the rapid evolution of Pt populations. The critical to control wheat leaf rust is to understand the pathogenicity mechanisms of Pt. Results: In this study, the spores of the Pt race PHNT (wheat leaf rust resistance gene Lr19-avirulent isolate) were mutagenized with ethyl methanesulfonate (EMS) and two Pt Lr19-virulent mutants named M1 and M2 were isolated, suggesting that they carry mutations affecting the Lr19-specific avirulent factor. RNA sequencing was performed on samples collected from the wheat cultivars Chinese Spring and TcLr19 that were infected by wild-type (WT) PHNT and the two Lr19-virulent mutant isolates at 14 days post-inoculation (dpi). The assembled transcriptome data were compared to the reference genome “Pt 1-1 BBBD Race 1.” A total of 216 differentially expressed genes (DEGs) were found from three different sample comparisons including M1-vs-WT, M2-vs-WT, and M1-vs-M2. Of these DEGs, 29 common DEGs were shared between M1-vs-WT and M2-vs-WT comparisons. Among the 216 DEGs encoding proteins, 30 were predicted to be effector candidates. Then 6 effector candidates (PTTG_27844, PTTG_05290, PTTG_27401, PTTG_27224, PTTG_26282, PTTG_25521) were verified that these genes were differentially expressed during Pt infection by quantitative real-time PCR (qRT-PCR) and were validated on tobacco, and the results showed that PTTG_27401 could inhibit progress of cell death (PCD) induced by BAX.Conclusions: Our results showed that a large number of genes participate in the interaction between Pt and TcLr19, which will provide valuable resources for the identification and targeting of AvrLr19 effector candidates and pathogenesis-related genes. Furthermore, our analyses are of great significance to reveal the pathogenesis of Pt.

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“…22) Purified recombinant isomalto-dextranase was prepared as described. 18) Glucoamylase from Rhizopus sp. was purchased from Wako Pure Chemical (Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae

Gozu

Ishizaki

Hosoyama

et al. 2016

Bioscience, Biotechnology, and Biochemistry

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Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

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Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27

Cited by 22 publications

References 51 publications

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporiumprovides insights into the mechanism of bypassing side chains

Transcriptome Analysis Based on Lr19-Virulent Mutants Strain Provides Clues for the Pathogenicity-Related Genes and Effectors of Puccinia Triticina

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae

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