The recognition mechanism of triple-helical β-1,3-glucan by a β-1,3-glucanase

Qin, Zhen; Yang, Dong; You, Xiao‐Zeng; Liu, Yu; Hu, Song-Qing; Yan, Qiaojuan; Yang, Shaoqing; Jiang, Zhengqiang

doi:10.1039/c7cc03330c

Cited by 33 publications

(34 citation statements)

References 24 publications

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“…8D). This mode of binding is suggestive of the triple helical structure of polymeric substrates (23). The groove observed in CbLPHases hints of a similar nature: it is quite wide and appears to accommodate higher-order structures of helical polymers (Fig.…”

Section: Resultsmentioning

confidence: 77%

“…8D). It has been shown that PbBgl64A acts primarily on helical polymer curdlan and less efficiently on branched polymers like laminarin (23). Both curdlan and laminarin are polysaccharides in which glucose molecules polymerize through ␤-1,3 bonding.…”

Section: Resultsmentioning

confidence: 99%

“…The probable function of such ancillary modules is to assist the enzymatic domain in substrate recognition and efficient binding (24). PbBgl64A, the LPHase from Paenibacillus barengoltzii, for which the crystal structure was reported recently (PDB ID 5H9Y) (23), is a two-domain protein, with a CBM56 domain in addition to the LPHase. The CBM56 moiety in PbBgl64A reportedly affects its enzyme activity, although the mechanism of its action is not known.…”

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confidence: 99%

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βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation

et al. 2019

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The prokaryotic βγ-crystallins are a large group of uncharacterized domains with Ca2+-binding motifs. We have observed that a vast number of these domains are found appended to other domains, in particular, the carbohydrate-active enzyme (CAZy) domains. To elucidate the functional significance of these prospective Ca2+ sensors in bacteria and this widespread domain association, we have studied one typical example from Clostridium beijerinckii, a bacterium known for its ability to produce acetone, butanol, and ethanol through fermentation of several carbohydrates. This novel glycoside hydrolase of family 64 (GH64), which we named glucanallin, is composed of a βγ-crystallin domain, a GH64 domain, and a carbohydrate-binding module 56 (CBM56). The substrates of GH64, β-1,3-glucans, are the targets for industrial biofuel production due to their plenitude. We have examined the Ca2+-binding properties of this protein, assayed its enzymatic activity, and analyzed the structural features of the β-1,3-glucanase domain through its high-resolution crystal structure. The reaction products resulting from the enzyme reaction of glucanallin reinforce the mixed nature of GH64 enzymes, in contrast to the prevailing notion of them being an exotype. Upon disabling Ca2+ binding and comparing different domain combinations, we demonstrate that the βγ-crystallin domain in glucanallin acts as a Ca2+ sensor and enhances the glycolytic activity of glucanallin through Ca2+ binding. We also compare the structural peculiarities of this new member of the GH64 family to two previously studied members. IMPORTANCE We have biochemically and structurally characterized a novel glucanase from the less studied GH64 family in a bacterium significant for fermentation of carbohydrates into biofuels. This enzyme displays a peculiar property of being distally modulated by Ca2+ via assistance from a neighboring βγ-crystallin domain, likely through changes in the domain interface. In addition, this enzyme is found to be optimized for functioning in an acidic environment, which is in line with the possibility of its involvement in biofuel production. Multiple occurrences of a similar domain architecture suggest that such a “βγ-crystallination”-mediated Ca2+ sensitivity may be widespread among bacterial proteins.

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Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation

et al. 2019

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“…The triple helical structure of β‐(1 → 3)‐glucan is stabilized by interstrand and intrastrand hydrogen bonds. The interstrand hydrogen bonds are formed between OH groups on C2 (C2OH), while intrastrand hydrogen bonds are formed between O4 and O5 of adjacent Glc subunits . In the Pro_7066 G6 complex, the C2OH groups are predominantly involved in the interaction with the SBS.…”

Section: Resultsmentioning

confidence: 99%

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

et al. 2019

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Glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐gluco‐oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far—glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X‐ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β‐(1 → 3)‐gluco‐oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.

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“…(2017) PDBID: 5EZJ Protein Structure Lin et al. (2017) PDBID: 5GZ4 Protein Structure Qin et al. (2017) PDBID: 5H9Y Protein Structure Lobner et al.…”

Section: Methodsmentioning

confidence: 99%

Automatically Fixing Errors in Glycoprotein Structures with Rosetta

et al. 2019

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Summary Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins which is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup and refinement of glycan-containing structures. We benchmarked this approach using twelve crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

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The recognition mechanism of triple-helical β-1,3-glucan by a β-1,3-glucanase

Cited by 33 publications

References 24 publications

βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation

βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

Automatically Fixing Errors in Glycoprotein Structures with Rosetta

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The recognition mechanism of triple-helical β-1,3-glucan by a β-1,3-glucanase

Cited by 33 publications

References 24 publications

βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca 2+ -Dependent Activity Modulation

βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca 2+ -Dependent Activity Modulation

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

Automatically Fixing Errors in Glycoprotein Structures with Rosetta

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βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation

βγ-Crystallination Endows a Novel Bacterial Glycoside Hydrolase 64 with Ca ²⁺ -Dependent Activity Modulation