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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 alpha-1,4-transferase and alpha-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 Asp363, 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 alpha-1,4-transferase activity and a reduced level of alpha-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 alpha-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.

SUBMITTER: Woo EJ

PROVIDER: S-EPMC2661413 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus.

Woo Eui-Jeon EJ Lee Seungjae S Cha Hyunju H Park Jong-Tae JT Yoon Sei-Mee SM Song Hyung-Nam HN Park Kwan-Hwa KH

The Journal of biological chemistry 20080814 42

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 alpha-1,4-transferase and alpha-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 Asp363, occupying subsites -1 to -3. Although generally similar to the mon ...[more]

PMID: 18703518

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Project description:BackgroundReverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle.ResultsFor the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C.ConclusionsThe two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.

Project description:Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than?80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel ?-N-acetylglucosaminidase (?-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an ?-GlcNAcase found a GlcNAc de-N-acetylase instead. The ?-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an ?-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricus IMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an ?-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the ?-glucosidase, catalyzed the hydrolysis of O-?-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

Dataset Information

Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus.

Publications

Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus.

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