Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

Kobayashi, Momoko; Saburi, Wataru; Nakatsuka, Daichi; Hondoh, Hironori; Kato, Kuniki; Okuyama, Masayuki; Mori, Haruhide; Kimura, Atsuo; Yao, Min

doi:10.1016/j.febslet.2015.01.005

Cited by 17 publications

(10 citation statements)

References 30 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The major conformation of Asp197 is consistent with previously reported structures of HPA-inhibitor complexes and appears to be stabilized by hydrogen bonding interactions with the C6 hydroxyl group of ECP [4,8,29]. This hydroxyl group is approximately in the same position as the endocyclic oxygen of the glucose moiety in the normal glycosyl enzyme intermediate, which has been seen in close proximity to the carbonyl oxygen of the catalytic nucleophile in several structures of GH13 glycosylenzymes [30][31][32]. The minor conformation of Asp197 is an unusual one for the nucleophile, with the v À1 angle relatively rotated by~20°toward Glu233 thereby reducing the distance between the carboxylic groups of Asp197 and Glu233 from 5.4…”

Section: Structural Analysis Of the Inactivated Enzymesupporting

confidence: 86%

Glucosyl epi‐cyclophellitol allows mechanism‐based inactivation and structural analysis of human pancreatic α‐amylase

et al. 2016

View full text Add to dashboard Cite

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic a-amylase we describe the chemoenzymatic synthesis of the disaccharide analog a-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic a-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.Keywords: activity-based probes; amylase; conduritol epoxide; GH13 structure; glycosyl enzyme; mechanism-based inhibition Digestion of starch in mammals starts with endocleavage of its a-1,4-glucosidic bonds by salivary and pancreatic a-amylases. This liberates oligosaccharides that are hydrolyzed further by a-glucosidases located on the gut wall, thereby delivering glucose to the bloodstream. Control of blood glucose levels can be achieved clinically by a-glucosidase inhibitors (AGI) such as acarbose and miglitol, which inhibit the gut wall enzymes [1,2]. However unpleasant side effects, in part associated with passage of the oligosaccharides to the lower gut, can limit patient compliance. An alternative strategy, which our laboratory has been exploring, is to employ specific inhibitors of the a-amylases that allow dietary oligosaccharides such as sucrose to be processed normally. This approach should minimize side effects while still reducing blood glucose levels. To that end, we have taken two approaches. High-throughput screening of natural product extracts is one approach, and has led us to the potent (K i = 8.1 nM) human pancreatic a-amylase (HPA) inhibitor montbretin A, which controls blood glucose in diabetic animals, and for which we have determined three-dimensional structures of its complex with HPA [3,4].The other approach has been through design of mechanism-based inhibitors that form covalent adducts with the HPA active site. While 2-deoxy-2-Abbreviations CP, cyclophellitol; ECP, epi-cyclophellitol; G-ECP, glucosyl epi-cyclophellitol; HPA, human pancreatic a-amylase; LaMalP, Lactobacillus acidophilus NCFM maltose phosphorylase.

show abstract

Section: Structural Analysis Of the Inactivated Enzymesupporting

confidence: 86%

Glucosyl epi‐cyclophellitol allows mechanism‐based inactivation and structural analysis of human pancreatic α‐amylase

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The reaction mechanism of -glucosidase has been studied for more than 30 years (Chiba, 1997;Davies & Henrissat, 1995). The glucosyl-enzyme intermediate of GH13 -glucosidase has been trapped by using its inactivator (McCarter & Withers, 1996), and structures of intermediates have been obtained by using general acid/base mutant enzymes with substrate analogues such as cyclodextrin glycosyltransferase from Bacillus circulans (BaCGTase; PDB entry 1cxl; Uitdehaag et al, 1999), NpAs (Jensen et al, 2004; PDB entry 1s46) and SmDG (Kobayashi et al, 2015;PDB entry 4wlc). In contrast to these structures obtained with inactive mutant enzymes and substrate analogues, the glucosyl-HaG structure strongly suggested the formation of a glucosyl-enzyme intermediate in its native form.…”

Section: Glucosyl-hag Intermediate Structurementioning

confidence: 99%

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Shen

Saburi

Gai

et al. 2015

Acta Crystallogr D Biol Crystallogr

Self Cite

View full text Add to dashboard Cite

α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long β → α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure.

show abstract

“…On the other hand, DGase exhibits high specificity for long chain substrates, such as dextran, and it also has transglycosylation activity . These differences in the specificities for substrate chain‐length may be partly accounted for by the differences in the shapes of the active sites . Among a number of substrates tested in this study, BmOGL and TlOGL were preferentially active on oligosaccharides bearing a α‐1,6‐glucosidic linkages, either between two glucose units (panose, isomaltose, and isomaltotriose) or between fructose and glucose moieties (isomaltulose) (Table ).…”

Section: Discussionmentioning

confidence: 82%

“…Generally, oligo‐1,6‐glucosidase prefers isomaltotriose, and hyrolyzes IMOs and dextran . On the other hand, DGase exhibits high specificity for long chain substrates, such as dextran, and it also has transglycosylation activity . These differences in the specificities for substrate chain‐length may be partly accounted for by the differences in the shapes of the active sites .…”

Section: Discussionmentioning

confidence: 99%

“…However, due to the slow hydrolysis of α‐1,6 glycosidic bonds in maltodextrins by glucoamylase during the saccharification process , short‐chain oligosaccharides consisted mainly of α‐1,6‐glucoside linkages (such as isomaltose, panose, isomaltotriose, isomaltotetraose, and other isomaltooligosaccharides [IMOs]) occur as by‐products , and D‐glucose yields higher than 96% of theoretical are therefore rarely achieved . Although isoamylase, pullulanase, dextran glucosidase and other debranching enzymes are capable of hydrolyzing these branched oligosaccharides, they only show preference for long‐chain substrates . Therefore, finding proper enzymes that can hydrolyze branched oligosaccharides of short lengths is of great importance for increasing the yield of glucose in starch processing industry.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular cloning and biochemical characterization of two novel oligo‐1,6‐glucosidases from Bacillus mycoides and Thermomyces lanuginosus

et al. 2017

View full text Add to dashboard Cite

High-glucose syrup has wide applications in the feed and fermentation industries. However, during its regular production process, because of the slow hydrolysis of a-1,6-glucosidic linkages in maltodextrins by glucoamylase, yields of glucose higher than 96% are rarely achieved. To find a suitable enzyme for the saccharification process, two novel genes encoding oligo-1,6-glucosidases (OGL, EC 3.2.1.10) from Bacillus mycoides (bmogl) and Thermomyces lanuginosus (tlogl) were successfully cloned and overexpressed in Pichia pastoris GS115. At the shake-flask fermentation level, the OGL activities of the two recombinants GS115 (pPIC9K-bmogl) and GS115 (pPIC9K-tlogl) were 994 and 1219 U/mL, respectively; and mature enzymes around 66-68 kDa were purified for characterization. Recombinant enzyme TlOGL exhibited higher thermostability than ever reported for OGLs, whereas BmOGL was stable under acidic pH, ranging from 4.0 to 8.0. Among the substrates tested, these two recombinant enzymes efficiently hydrolyzed isomaltose and isomaltotriose, but had no activity against maltose and maltotriose. Besides these characteristics, nearly complete hydrolysis of isomaltotriose and 90% of isomaltooligosaccharides (IMOs) into glucose was also observed for them, which makes them good candidates for subsequent use in improving the yield of glucose from starch. To the best of our knowledge, this is the first report on the expression and characterization of OGLs from B. mycoides and T. lanuginosus.

show abstract

Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate‐binding site in GH13 dextran glucosidase

Cited by 17 publications

References 30 publications

Glucosyl epi‐cyclophellitol allows mechanism‐based inactivation and structural analysis of human pancreatic α‐amylase

Glucosyl epi‐cyclophellitol allows mechanism‐based inactivation and structural analysis of human pancreatic α‐amylase

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Molecular cloning and biochemical characterization of two novel oligo‐1,6‐glucosidases from Bacillus mycoides and Thermomyces lanuginosus

Contact Info

Product

Resources

About