Crystal structure of the β-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability

120

The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

Section: Methodsmentioning

confidence: 99%

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Kim

Lee

Warren

et al. 2004

120

“…Our simulations were based on the crystal structure of Ss␤gly refined at 2.6 Å (Protein Data Bank entry 1GOW) (22).…”

Section: Molecular Mass Estimation By Ferguson Analysis Of Linear Tramentioning

confidence: 99%

Thermal Stability and Aggregation of Sulfolobus solfataricus β-Glycosidase Are Dependent upon the N-∈-Methylation of Specific Lysyl Residues

Febbraio

Andolfo

Tanfani

et al. 2004

Self Cite

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of ␤-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native ␤-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of ␤-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of ␤-glycosidase from S. solfataricus.

“…The only examples of glycosyl hydrolases characterized in this genus are a secreted ␣-amylase, an intracellular ␣-glucosidase from S. solfataricus strain 98/2 (17,18), and a ␤-glycosidase that has been purified from strains MT4 and P2 (19,20). The two ␣-glycosyl hydrolases are involved in the utilization of dextrins, whereas the function in vivo of the ␤-glycosidase (LacS), which is under active study in our laboratory, is still obscure (21)(22)(23). In an effort to determine the full set of glycosyl hydrolases produced by this hyperthermophilic Archaeon, we have identified a novel ␣-xylosidase (XylS) with high specificity for isoprimeverose and xyloglucan oligosaccharides.…”

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

“…Surprisingly, the analysis of a 15-kilobase (kb) sequenced region, which comprises xylS, revealed that it mapped in the locus of the lacS gene encoding for the ␤-glycosidase that has been extensively studied in our laboratory (19,(21)(22)(23). As reported previously, lacS is preceded, in the same orientation, by a gene encoding a putative membrane protein homologous to the Major Facilitator Superfamily (mfs) (30) and followed, in the opposite orientation, by a 1785-bp open reading frame (orf3) encoding a 592-amino acid polypeptide of unknown function.…”

Section: Identification and Sequence Analysis Of Xyls And Its Flankingmentioning

confidence: 99%

Identification and Molecular Characterization of the First α-Xylosidase from an Archaeon

Moracci¹,

Cobucci‐Ponzano²,

Trincone³

et al. 2000

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We here report the first molecular characterization of an ␣-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80°C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to ␣-glucosidases, ␣-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a ␤-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The ␣-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90°C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the ␣-xylosidase and the ␤-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.