大家好，今天推送的文章是2015年5月发表在 Acta crystallographica. Section D, Structural biology 上的 “Structural analysis of the a-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism”，通讯作者为北海道大学先进生命科学学院的 姚闵 教授。
α-glucosidase, which catalyzes the hydrolysis of the non-reducing end of the substrate α-glucosididic bonds, is important for the metabolism of α-glucoside. Mininomonas H11 α-glucosidase (HaG) belongs to the glycoside hydrolase family 13 (GH13) and has high hydrolytic activity against only α-(1→4)-linked disaccharide maltose in native substrates. Although several three-dimensional structures of GH13 members have been resolved, the disaccharide specificity and α-(1→4) recognition mechanism of α-glucosidase are unclear due to the lack of corresponding substrate-binding structures.
In this study, the authors' team addressed four crystal structures of HaG: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant complexed with its native substrate maltose, and the D202N mutant complex with D-glucose and glycerol, revealing new insights into the substrate specificity and catalytic mechanisms of α-glucosidase.
1
Overall structure and active site of HaG
All four crystal structures contain dimers in asymmetric units. Similar to the GH13 family members, the structure of HaG consists of a main catalytic domain A (residues 1-106 and 175-465), a ring-rich domain B (residues 107-174), and a highly conserved domain C (residues 466-538) formed by two antiparallel β-folds (Figure 1a).
Domain A is the catalytic domain, formed by the classical TIM barrel, sandwiched between domains B and C.
Domain B contains 1 α-helix and 3 β-chains, which extend from domain A and together with domain A forms the wall of the catalytic pocket. Domain C appears to stabilize the conformation of the entire structure, but its function remains unclear. In HaG, there are four phenylalanines (Phe479, Phe492, Phe516, and Phe534) in the C domain, all of which are located on the surface in contact with the A domain, forming a hydrophobic interface that stabilizes the entire structure.
The overall structure of HaG has the highest similarity with the sucrose isomerase MutB (PDB: 2pwh) from Pseudomonas mesophila MX-45 in GH13 family members, with a root mean square deviation of 1.9 Å. While the structure of the sub-site -1 at the bottom of the pocket of the two enzyme activity sites is similar, the subsite +1 is different (Figure 1B). In addition, Phe256 and Phe280 (MutB_Phe256 and MutB_Phe280) in MutB are conserved in sucrose isomerase and are essential for controlling intramolecular transglucosylation, but in HaG the corresponding residues of MutB_Phe256, Gly273 do not interact with substrates. Phe297 of HaG corresponds to MutB_Phe280 and is further away from the substrate in the HaG-substrate composite structure. This structural feature may be reflected in their transglycosylation specificity.
The active site pocket of HaG consists of 15 residues. Eight residues, including the catalytic nucleophile Asp202, the general acid/base catalyst Glu271, Asp333, Arg200, His105, His332, Phe297 and Tyr65, were conserved in the GH13 family. Six residues, Asp62, Arg400, Phe166, Thr203, Phe206, and Phe147, are conserved among the homologs of HaG, and another residue, Gly228, is found only in HaG. The distance between the catalytic nucleophile (Asp202) and the general acid/base catalyst (Glu271) is 5.6Å, a phenomenon that is conserved in other glycosidases. The ring (Leu201–Arg246) is located between the fourth α-sheet and the α-helix (β→α loop 4) of the HaG catalytic A domain, longer than in the other GH13 members (Figure 1c), and it is located just above the subsite +1 glucose moiety, covering the main part of the active site entrance (Figure 1b)
The conformations of the above active sites are very similar across all four structures (root mean square error of 15 Cα atoms within 0.21 Å), which means that the reaction does not require the binding residues of HaG or the reaction residues undergo a conformational change. Indicates that HaG requires less energy to initiate a catalytic reaction than other enzymes.
Figure 1
2
Structure of glucosyl-HaG intermediates
The authors' team mutated the common acid/base Glu271 to inactive Gln271 (E271Q) in order to obtain a substrate-binding complex. This residue is located at sublocus +1 and therefore does not affect substrate binding at sublocus -1. The glucose residue at sublosite-1 is pinned in place by six conserved residues: His105, Asp62, Arg200, Arg400, Asp333, and His332 (Figure 2b). A salt bridge forms between Asp62 and Arg400, which is important for the identification of glucose residues at the non-reducing end. The team of authors found that two residues, Thr203 and Phe297, were associated with glycosidic bond recognition. Thr203 is hypothesized to be one of the important residues for linkage specificity. α-(1→4)-specific glucosidase generally has Thr or Ala at this location, whereas α-(1→6)-specific glucosidase has Val. The superposition of E271Q-Mal with isomaltase from Saccharomyces cerevisiae with isomaltose complex (PDB:3axh) showed that the reduced end of maltose could not bind to the +1 subsite of isomaltase due to steric hindrance from Val216, whereas Thr203 of HaG left sufficient space for maltose and isomaltose (Figure 3a).
Figure 2
Conversely, HaG's Phe297 can hinder the binding of isomaltose to HaG, binding to maltose. In isomaltase, phenylalanine (Phe303) at the same location adopts a different conformation than Phe297 in HaG (Figure 3a), which forms the inner wall of the subsite +1 through hydrophobic interaction with Phe206, Ile146, and Phe166. The O atom of another non-conserved residue, Gly228, is located in the middle of β→αloop 4 and interacts with maltose (Figure 2b). In dextran glucosidase, Lys275 and Glu371 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at the +1 subsite, whereas in HaG, only Gly228 is located at the +1 subsite within the hydrogen bond interaction distance between the substrate and the enzyme, which has an auxiliary function in substrate binding and recognizes only α-maltose. The superposition of E271Q-Mal and MutB-sucrose (PDB:2pwe) suggests a significant twist of the glucose residue at the +1 subsite between maltose and sucrose, resulting in Gly228 being closer to maltose than to sucrose (Fig.3b), which may be related to selective activity against maltose, as sucrose is a poor substrate for HaG. Phe297 may also result in lower activity of HaG against sucrose due to space constraints (2.6 Å).
Figure 3
3
D202N-Glc-Gol complex
HaG crystals have a diffraction resolution of 1.47 Å and are grown in the presence of sucrose. The Fo-Fc electron density plot clearly shows that by interacting with Asp202, the glucose-based moiety is trapped in subsite-1 (Figure 6), indicating that the reaction intermediate state is captured in this complex structure. The glucosylase intermediate of GH13α-glucosidase has been captured by using its inactivator, and the structure of the intermediate has been obtained by using a universal acid/base mutant enzyme with a substrate analogue such as cyclodextrin glycosyltransferase.
Compared to these structures obtained with inactive mutant enzymes and substrate analogues, the glucosyl-HaG structure strongly suggests that its native form forms a glucosyl-enzyme intermediate. The authors speculate that the intermediate is trapped in the crystal for the following reasons: (i) the rate of deglycosylation is very slow in the absence of monovalent cations, (ii) sucrose is a poor substrate for HaG, and (iii) the crystallization buffer does not contain monovalent cations at pH 7.5, which is above the optimal pH (6.5). The glucosyl ring is tightly fixed to the non-reducing end of the -1 subsite in E271Q-Mal and adopts the 4C1 chair conformation (Figure 6), with no significant conformational changes observed compared to HaG. In the E271Q-Mal structure, the distance between the C1 of the glucosyl ring and the O of Asp202 is 3.1Å, which is much longer than in the glucosyl-HaG intermediate. This suggests that the C2-C1-O5-C5 twist angle of the glucosyl ring is altered to achieve a catalytic reaction.
Figure 6
The inactive D202N mutant structure complexed with a glucose molecule and a glycerol molecule (D202N-Glc-Gol) enables the study of the transglycosylation process for the production of α-D-glucosylglycerol (αGG; Figure 7a), and the conformation of the proteins in the D202NGlc-Gol complex is almost identical to that of HaG. Glucose occupies subsite -1, while glycerol appears at subsite +1 (Figure 7b). Almost identical to the E271Q-Mal structure. The C2-C1-O5-C5 twist angle of glucose in D202N-Glc-Gol is 65.7°, which is similar to that of the glucosyl ring in the glucosyl-HaG intermediate. However, there is a slight rotation between the glucose residues in D202N-Glc-Gol and the glucose residues in the glucosyl-HaG intermediate, as the distance between the two C1 atoms is 0.9 Å (Figure 7c), and the glucosyl ring will move away from the catalytic site after the hydrolysis reaction.
Figure 7
In the present study, the authors' team resolved four crystal structures of HaG: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant complexed with its native substrate maltose, and the D202N mutant complex with d-glucose and glycerol. These structures unambiguously provide insights into the substrate specificity and catalytic mechanisms of HaG. The peculiar β→α loop 4, present in α-glucosidase, is responsible for the strict recognition of disaccharides due to steric hindrance. Two residues, Thr203 and Phe297, were found with the assistance of Gly228 to determine the glycosidic bond specificity of the substrate at subsite +1.
Article Information:
http://dks.doi.org/10.1107/s139900471500721s