Structural Insight into the Bifunctional Mechanism of the Glycogen-debranching Enzyme TreX from the Archaeon Sulfolobus solfataricus

Abstract

TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for ␣-1,4-transferase and ␣-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp 363 , occupying subsites ؊1 to ؊3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the "flexible loop" (amino acids 399 -416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in ␣-1,4-transferase activity and a reduced level of ␣-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99 -97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the ␣-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.Debranching enzymes are divided into two groups. One includes isoamylase and pullulanase found in bacteria and plants, and the other includes glycogen-debranching enzymes (GDEs) 4 found in mammals and yeast. Whereas the former has only a single ␣-1,6-glycosidic bond hydrolyzing activity for glycogen and amylopectin, the GDEs in eukaryotes are bifunctional, possessing both ␣-1,4-transferase (EC 2.4.1.25) and ␣-1,6-glucosidase (EC 3.2.1.33) activities within a single polypeptide chain (1, 2). The structure of GDE in eukaryotes has yet to be determined.TreX is an archaeal enzyme in Sulfolobus that debranches the side chain of glycogen into maltodextrin, which is further converted to trehalose (3). It belongs to the GH13 (glycoside hydrolase 13) family and shows high sequence similarity to isoamylase and pullulanase. The amino acid sequence of TreX shows 74% homology to isoamylase from Sulfolobus acidocaldarium, 42% homology to the glycogen operon protein GlgX from Escherichia coli, and 30% homology to isoamylase from Pseudomonas.However, despite its high sequence similarity to isoamylase, which has only a single ␣-1,6-glycosidic bond hydrolyzing activity, TreX exhibits ␣-1,4-transferase as well as ␣-1,6-glucosidase activity, similar to GDEs in mammals and yeast (4, 5). To date, it is the only GDE found in bacteria/archaea with ␣-1,4-transferase activity, transferring the ␣-1,4-glucan oligosaccharides from one molecule to another using various substrates, including glycogen and amylopectin (4).In a previous study, we cloned th...

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

“…Unexpectedly, TreX was active on branched chains of DP2 to DP10; transfer products were evident with all tested substrates. This result agrees with the suggestion of Woo et al (21) that subunit tetramerization generates a channel-like cavity that allows long branch chains to be accommodated. An isoamylase from Pseudomonas spp.…”

“…However, our previous study showed that TreX exhibits both hydrolysis and transglycosylation activities by catalyzing the breakage and transfer of ␣-1,4-glucan oligosaccharides among chains (20). We also obtained a three-dimensional structure of TreX and identified two distinct active site configurations, in line with the bifunctional nature of the enzyme (21). The bifunctional debranching enzymes from mammalian tissues transfer the maltotetraosyl moiety to the nonreducing end of glucose, elongating the branch chain with an ␣-1,4-glycosidic linkage that is immediately hydrolyzed by glycogen phosphorylase (GlgP).…”

“…As shown in Fig. 5C, comparison of the chromatographic profile with that of (Glc 2 ) 2 -␤-CD reference materials (21) indicated that the transfer product was a mixture of 6 1 ,6 3 -and 6 1 ,6 4 -(Glc 4 ) 2 -␤-CD at a molar ratio close to unity. Additionally, the formation of 6 1 ,6 4 -(Glc 4 ) 2 -␤-CD observed in the present study and the synthesis of 6 1 ,6 4 -(Glc 2 ) 2 -␤-CD reported by Kang et al (26) support the idea that the transfer products produced from branch chains varying widely in size are similar.…”

Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

“…Precision-indication merging R factor values (R pim ) (26) calculated using Aimless (27), along with data completeness and CC 1/2 (28) were considered in the determination of high-resolution cutoffs used for structural refinement. The CrISA1 structure was determined by a molecular replacement method using Phaser (29) and a CHAINSAW (30) trimmed structure of glycogen debranching enzyme TreX (PDB code 2VNC) as a search model (31). Two molecules were found in the asymmetric unit, which corresponded to a solvent content of 65%.…”

Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase from Staphylothermus marinus

“…The only exception is represented by the ␤-subunit of AMPK (37), which showed that CBM48 is a separate domain that binds cyclodextrin to increase its binding ability for the glucosyl polymeric structures commonly found in glycogen (62). Despite the wealth of structural information on CBM48 (36,52,55,(63)(64)(65)(66)(67) (for a review, see Ref. 68)), this paper is, to the best of our knowledge, the first to demonstrate that an independent CBM domain folds to interact with the catalytic domain and participates in substrate binding at the active site.…”